Δ-9 elongases and their use in making polyunsaturated fatty acids

ABSTRACT

The present invention relates to Δ9 elongases, which have the ability to convert linoleic acid (LA; 18:2 ω-6) to eicosadienoic acid (EDA; 20:2 ω-6) and/or α-linolenic acid (ALA; 18:3 ω-3) to eicosatrienoic acid (ETrA; 20:3 ω-3). Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding Δ9 elongases along with a method of making long-chain polyunsaturated fatty acids (PUFAs) using these Δ9 elongases in oleaginous yeast are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division application of U.S. patent applicationSer. No. 12/102,879 filed Apr. 15, 2008, granted as U.S. Pat. No.7,794,701 on Sep. 14, 2010, which claims the benefit of U.S. ProvisionalApplication No. 60/911,925, filed Apr. 16, 2007, now abandoned, theentire contents of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to the identification of polynucleotide sequencesencoding Δ9 fatty acid elongases and the use of these elongases inmaking long-chain polyunsaturated fatty acids (PUFAs).

BACKGROUND OF THE INVENTION

Today, a variety of different hosts including plants, algae, fungi,stramenopiles and yeast are being investigated as means for commercialPUFA production. Genetic engineering has demonstrated that the naturalabilities of some hosts (even those natively limited to linoleic acid(LA; 18:2 ω-6) and α-linolenic acid (ALA; 18:3 ω-3) fatty acidproduction) can be substantially altered to result in high-levelproduction of various long-chain ω-3/ω-6 PUFAs. Whether this is theresult of natural abilities or recombinant technology, production ofarachidonic acid (ARA; 20:4 ω-6), eicosapentaenoic acid (EPA; 20:5 ω-3)and docosahexaenoic acid (DHA; 22:6 ω-3) may require expression of a Δ9elongase.

Most Δ9 elongase enzymes identified so far have the ability to convertboth LA to eicosadienoic acid (EDA; 20:2 ω-6) and ALA to eicosatrienoicacid (ETrA; 20:3 ω-3) (wherein dihomo-γ-linolenic acid (DGLA; 20:3 ω-6)and eicosatetraenoic acid (ETA; 20:4 ω-3) are subsequently synthesizedfrom EDA and ETrA, respectively, following reaction with a Δ8desaturase; ARA and EPA are subsequently synthesized from DGLA and ETA,respectively, following reaction with a Δ5 desaturase; and, DHAsynthesis requires subsequent expression of an additional C_(20/22)elongase and a Δ4 desaturase).

In spite of the need for new methods for the production of ARA, EPA andDHA, few Δ9 elongase enzymes have been identified. A Δ9 elongase fromIsochrysis galbana is publicly available (described in GenBank AccessionNo. AAL37626, as well as PCT Publications No. WO 02/077213, No. WO2005/083093, No. WO 2005/012316 and No. WO 2004/057001). PCTPublications No. WO 2007/061845 and No. WO 2007/061742 (Applicants'Assignee's co-pending applications), disclose Δ9 elongases from Euglenagracilis and Eutreptiella sp. CCMP389, as well as Δ9 elongase motifs.

Thus, there is need for the identification and isolation of additionalgenes encoding Δ9 elongases that will be suitable for heterologousexpression in a variety of host organisms for use in the production ofω-3/ω-6 fatty acids.

Applicants have solved the stated problem by isolating genes encoding Δ9fatty acid elongases from Euglena anabaena.

SUMMARY OF THE INVENTION

The present invention relates to new genetic constructs encodingpolypeptides having Δ9 elongase activity, and their use in algae,bacteria, yeast, euglenoids, stramenopiles and fungi for the productionof PUFAs. Accordingly the invention provides a microbial host cellcomprising an isolated polynucleotide comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having Δ9        elongase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:13 or SEQ ID NO:14;    -   (b) a nucleotide sequence encoding a polypeptide having 49        elongase activity, wherein the nucleotide sequence has at least        80% sequence identity, based on the BLASTN method of alignment,        when compared to a nucleotide sequence as set forth in SEQ ID        NO:11, SEQ ID NO:12 or SEQ ID NO:26;    -   (c) a nucleotide sequence encoding a polypeptide having Δ9        elongase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence as set forth        in SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:26; or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

In another embodiment the invention provides a method for the productionof eicosadienoic acid comprising:

-   -   a) providing a microbial host cell comprising:        -   (i) a recombinant nucleotide molecule encoding a Δ9 elongase            polypeptide having at least 80% amino acid identity, based            on the Clustal V method of alignment, when compared to an            amino acid sequence as set forth in SEQ ID NO:13 or SEQ ID            NO:14; and,        -   (ii) a source of linoleic acid;    -   b) growing the microbial host cell of step (a) under conditions        wherein the nucleic acid fragment encoding the Δ9 elongase        polypeptide is expressed and the linoleic acid is converted to        eicosadienoic acid; and,    -   c) optionally recovering the eicosadienoic acid of step (b).

In an additional embodiment the invention provides a method for theproduction of eicosatrienoic acid comprising:

-   -   a) providing a microbial host cell comprising:        -   (i) a recombinant nucleotide molecule encoding a Δ9 elongase            polypeptide having at least 80% amino acid identity, based            on the Clustal V method of alignment, when compared to an            amino acid sequence as set forth in SEQ ID NO:13 or SEQ ID            NO:14; and,        -   (ii) a source of α-linolenic acid;    -   b) growing the microbial host cell of step (a) under conditions        wherein the nucleic acid fragment encoding the Δ9 elongase        polypeptide is expressed and the α-linolenic acid is converted        to eicosatrienoic acid; and,    -   c) optionally recovering the eicosatrienoic acid of step (b).

In another embodiment the invention provides an isolated nucleic acidmolecule which encodes a Δ9 elongase as set forth in SEQ ID NO:26wherein at least 98 codons are codon-optimized for expression inYarrowia sp.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

FIG. 1 is a representative ω-3 and ω-6 fatty acid biosynthetic pathwayproviding for the conversion of myristic acid through variousintermediates to DHA.

FIG. 2 shows a chromatogram of the lipid profile of an Euglena anabaenacell extract as described in the Examples.

FIG. 3 provides plasmid maps for the following: (A) pY115 (SEQ IDNO:19); (B) pY159 (SEQ ID NO:23); (C) pY173 (SEQ ID NO:24); and, (D)pY174 (SEQ ID NO:25).

FIGS. 4A and 4B show a comparison of the nucleotide sequences of EaD9Elo(SEQ ID NO:11) and EaD9ES (SEQ ID NO:26).

FIG. 5 provides plasmid maps for the following: (A) pEaD9ES (SEQ IDNO:28); and, (B) pZUFmEaD9eS (SEQ ID NO:29).

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1-4, 9-19 and 22-29 are ORFs encoding genes or proteins (orportions thereof), or plasmids, as identified in Table 1.

TABLE 1 Summary Of Nucleic Acid And Protein SEQ ID Numbers Nucleic acidSEQ Protein Description and Abbreviation ID NO. SEQ ID NO. Euglenaanabaena Δ9 elongase cDNA 1 — sequence (“EaD9Elo1”) (1129 bp) Euglenaanabaena Δ9 elongase cDNA 2 — sequence (“EaD9Elo2”) (1145 bp) Euglenagracilis Δ9 elongase coding sequence 3 4 (“EgD9e”)  (774 bp) (258 AA)Plasmid pLF121-1 9 — (3668 bp) Plasmid pLF121-2 10 — (3684 bp) Euglenaanabaena Δ9 elongase coding 11 13 sequence (“EaD8Des1 CDS” or“EaD9Elo1”,  (774 bp) (258 AA) respectively) Euglena anabaena Δ9elongase coding 12 14 sequence (“EaD8Des2 CDS” or “EaD9Elo2”,  (774 bp)(258 AA) respectively) Plasmid pKR906 15 — (4311 bp) Isochrysis galbanaΔ9 elongase (IgD9e) — 16 (263 AA) Plasmid pDMW263 17 — (9472 bp) PlasmidpDMW237 18 — (7879 bp) Plasmid pY115 19 — (7783 bp) Plasmid pY158 22 —(6992 bp) Plasmid pY159 23 — (8707 bp) Plasmid pY173 24 — (8219 bp)Plasmid pY174 25 (8235 bp) Synthetic Δ9 elongase, derived from Euglena26 27 anabaena, codon-optimized for expression in  (774 bp) (258 AA)Yarrowia lipolytica (“EaD9eS”) Plasmid pEaD9ES 28 (3497 bp) PlasmidpZUFmEaD9eS 29 — (7769 bp)

SEQ ID NOs:5 and 6 correspond to oligonucleotides oEugEL1-1 andoEugEL1-2, respectively, used for amplification of the Euglena gracilisΔ9 elongase.

SEQ ID NOs:7 and 8 correspond to the M13F universal primer and primerM13-28Rev, respectively, used for end-sequencing of Euglena anabaena DNAinserts.

SEQ ID NOs:20 and 21 correspond to primers oYFBA1 and oYFBA1-6,respectively, used to amplify the FBAINm promoter from plasmid pY115.

DETAILED DESCRIPTION OF THE INVENTION

New Euglena anabaena Δ9 elongase enzymes and genes encoding the samethat may be used for the manipulation of biochemical pathways for theproduction of healthful PUFAs are disclosed herein.

PUFAs, or derivatives thereof, are used as dietary substitutes, orsupplements, particularly infant formulas, for patients undergoingintravenous feeding or for preventing or treating malnutrition.Alternatively, the purified PUFAs (or derivatives thereof) may beincorporated into cooking oils, fats or margarines formulated so that innormal use the recipient would receive the desired amount for dietarysupplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements or other food products and may finduse as anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use (human or veterinary).

DEFINITIONS

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

“Triacylglycerols” are abbreviated TAGs.

The term “invention” or “present invention” as used herein is not meantto be limiting to any one specific embodiment of the invention butapplies generally to any and all embodiments of the invention asdescribed in the claims and specification.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

The term “fatty acids” refers to long-chain aliphatic acids (alkanoicacids) of varying chain lengths, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. Additional details concerning thedifferentiation between “saturated fatty acids” versus “unsaturatedfatty acids”, “monounsaturated fatty acids” versus “polyunsaturatedfatty acids” (or “PUFAs”), and “omega-6 fatty acids” (ω-6 or n-6) versus“omega-3 fatty acids” (ω-3 or n-3) are provided in U.S. Pat. No.7,238,482.

Fatty acids are described herein by a simple notation system of “X:Y”,where X is the total number of carbon (C) atoms in the particular fattyacid and Y is the number of double bonds. The number following the fattyacid designation indicates the position of the double bond from thecarboxyl end of the fatty acid with the “c” affix for thecis-configuration of the double bond (e.g., palmitic acid (16:0),stearic acid (18:0), oleic acid (18:1, 9c), petroselinic acid (18:1,6c), LA (18:2, 9c,12c), GLA (18:3, 6c,9c,12c) and ALA (18:3,9c,12c,15c)). Unless otherwise specified, 18:1, 18:2 and 18:3 refer tooleic, LA and ALA fatty acids, respectively. If not specifically writtenas otherwise, double bonds are assumed to be of the cis configuration.For instance, the double bonds in 18:2 (9,12) would be assumed to be inthe cis configuration.

Nomenclature used to describe PUFAs in the present disclosure is shownbelow in Table 2. In the column titled “Shorthand Notation”, theomega-reference system is used to indicate the number of carbons, thenumber of double bonds and the position of the double bond closest tothe omega carbon, counting from the omega carbon (which is numbered 1for this purpose). The remainder of the Table summarizes the commonnames of ω-3 and ω-6 fatty acids and their precursors, the abbreviationsthat will be used throughout the remainder of the specification, andeach compounds' chemical name.

TABLE 2 Nomenclature of Polyunsaturated Fatty Acids and PrecursorsShorthand Common Name Abbreviation Chemical Name Notation Myristic —tetradecanoic 14:0 Palmitic PA or hexadecanoic 16:0 PalmitatePalmitoleic — 9-hexadecenoic 16:1 Stearic — octadecanoic 18:0 Oleic —cis-9-octadecenoic 18:1 Linoleic LA cis-9,12-octadecadienoic 18:2 ω-6γ-Linolenic GLA cis-6,9,12- 18:3 ω-6 octadecatrienoic Eicosadienoic EDAcis-11,14-eicosadienoic 20:2 ω-6 Dihomo-γ- DGLAcis-8,11,14-eicosatrienoic 20:3 ω-6 linolenic Sciadonic SCIcis-5,11,14-eicosatrienoic 20:3b ω-6 Arachidonic ARA cis-5,8,11,14- 20:4ω-6 eicosatetraenoic α-Linolenic ALA cis-9,12,15- 18:3 ω-3octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 ω-3octadecatetraenoic Eicosatrienoic ETrA or ERA cis-11,14,17- 20:3 ω-3eicosatrienoic Eicosa- ETA cis-8,11,14,17- 20:4 ω-3 tetraenoiceicosatetraenoic Juniperonic JUP cis-5,11,14,17- 20:4b ω-3eicosatrienoic Eicosa- EPA cis-5,8,11,14,17- 20:5 ω-3 pentaenoiceicosapentaenoic Docosatrienoic DRA cis-10,13,16- 22:3 ω-6docosatrienoic Docosa- DTA cis-7,10,13,16- 22:4 ω-6 tetraenoicdocosatetraenoic Docosa- DPAn-6 cis-4,7,10,13,16- 22:5 ω-6 pentaenoicdocosapentaenoic Docosa- DPA cis-7,10,13,16,19- 22:5 ω-3 pentaenoicdocosapentaenoic Docosa- DHA cis-4,7,10,13,16,19- 22:6 ω-3 hexaenoicdocosahexaenoic

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

“Percent (%) PUFAs in the total lipid and oil fractions” refers to thepercent of PUFAs relative to the total fatty acids in those fractions.The term “total lipid fraction” or “lipid fraction” both refer to thesum of all lipids (i.e., neutral and polar) within an oleaginousorganism, thus including those lipids that are located in thephosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fractionand triacylglycerol (TAG or oil) fraction. However, the terms “lipid”and “oil” will be used interchangeably throughout the specification.

A metabolic pathway, or biosynthetic pathway, in a biochemical sense,can be regarded as a series of chemical reactions occurring within acell, catalyzed by enzymes, to achieve either the formation of ametabolic product to be used or stored by the cell, or the initiation ofanother metabolic pathway (then called a flux generating step). Many ofthese pathways are elaborate, and involve a step by step modification ofthe initial substance to shape it into a product having the exactchemical structure desired.

The term “PUFA biosynthetic pathway” refers to a metabolic process thatconverts oleic acid to ω-6 fatty acids such as LA, EDA, GLA, DGLA, ARA,DRA, DTA and DPAn-6 and ω-3 fatty acids such as ALA, STA, ETrA, ETA,EPA, DPA and DHA. This process is well described in the literature(e.g., see PCT Publication No. WO 2006/052870). Simplistically, thisprocess involves elongation of the carbon chain through the addition ofcarbon atoms and desaturation of the molecule through the addition ofdouble bonds, via a series of special desaturation and elongationenzymes (i.e., “PUFA biosynthetic pathway enzymes”) present in theendoplasmic reticulim membrane. More specifically, “PUFA biosyntheticpathway enzyme” refers to any of the following enzymes (and genes whichencode said enzymes) associated with the biosynthesis of a PUFA,including: a Δ9 elongase, a C_(14/16) elongase, a C_(16/18) elongase, aC_(18/20) elongase, a C_(20/22) elongase, a Δ4 desaturase, a Δ5desaturase, a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17desaturase, a Δ9 desaturase and/or a Δ8 desaturase.

The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set ofgenes which, when expressed under the appropriate conditions encodeenzymes that catalyze the production of either or both ω-3 and ω-6 fattyacids. Typically the genes involved in the ω-3/ω-6 fatty acidbiosynthetic pathway encode PUFA biosynthetic pathway enzymes. Arepresentative pathway is illustrated in FIG. 1, providing for theconversion of myristic acid through various intermediates to DHA, whichdemonstrates how both ω-3 and ω-6 fatty acids may be produced from acommon source. The pathway is naturally divided into two portions whereone portion will generate ω-3 fatty acids and the other portion, ω-6fatty acids.

The term “functional” as used herein in context with the ω-3/ω-6 fattyacid biosynthetic pathway means that some (or all) of the genes in thepathway express active enzymes, resulting in in vivo catalysis orsubstrate conversion. It should be understood that “ω-3/ω-6 fatty acidbiosynthetic pathway” or “functional ω-3/ω-6 fatty acid biosyntheticpathway” does not imply that all the PUFA biosynthetic pathway enzymegenes are required, as a number of fatty acid products will only requirethe expression of a subset of the genes of this pathway.

The term “Δ6 desaturase/Δ6 elongase pathway” will refer to a PUFAbiosynthetic pathway that minimally includes at least one Δ6 desaturaseand at least one C_(18/20) elongase (also referred to as a Δ6 elongase),thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA,respectively, with GLA and/or STA as intermediate fatty acids. Withexpression of other desaturases and elongases, ARA, EPA, DPA and DHA mayalso be synthesized.

The term “Δ9 elongase/Δ8 desaturase pathway” will refer to a PUFAbiosynthetic pathway that minimally includes at least one Δ9 elongaseand at least one Δ8 desaturase, thereby enabling biosynthesis of DGLAand/or ETA from LA and ALA, respectively, with EDA and/or ETrA asintermediate fatty acids. With expression of other desaturases andelongases, ARA, EPA, DPA and DHA may also be synthesized.

The term “intermediate fatty acid” refers to any fatty acid produced ina fatty acid metabolic pathway that can be further converted to anintended product fatty acid in this pathway by the action of othermetabolic pathway enzymes. For instance, when EPA is produced using theΔ9 elongase/Δ8 desaturase pathway, EDA, ETrA, DGLA, ETA and ARA can beproduced and are considered “intermediate fatty acids” since these fattyacids can be further converted to EPA via action of other metabolicpathway enzymes.

The term “by-product fatty acid” refers to any fatty acid produced in afatty acid metabolic pathway that is not the intended fatty acid productof the pathway nor an “intermediate fatty acid” of the pathway. Forinstance, when EPA is produced using the Δ9 elongase/Δ8 desaturasepathway, sciadonic acid (SCI) and juniperonic acid (JUP) also can beproduced by the action of a Δ5 desaturase on either EDA or ETrA,respectively. They are considered to be “by-product fatty acids” sinceneither can be further converted to EPA by the action of other metabolicpathway enzymes.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a fattyacid or precursor of interest. Despite use of the omega-reference systemthroughout the specification to refer to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system.Desaturases of interest include, for example: (1) Δ8 desaturases thatdesaturate a fatty acid between the eighth and ninth carbon atomnumbered from the carboxyl-terminal end of the molecule and that can,for example, catalyze the conversion of EDA to DGLA and/or ETrA to ETA;(2) Δ5 desaturases that catalyze the conversion of DGLA to ARA and/orETA to EPA; (3) Δ6 desaturases that catalyze the conversion of LA to GLAand/or ALA to STA; (4) Δ4 desaturases that catalyze the conversion ofDPA to DHA and/or DTA to DPAn-6; (5) Δ12 desaturases that catalyze theconversion of oleic acid to LA; (6) Δ15 desaturases that catalyze theconversion of LA to ALA and/or GLA to STA; (7) Δ17 desaturases thatcatalyze the conversion of ARA to EPA and/or DGLA to ETA; and, (8) Δ9desaturases that catalyze the conversion of palmitic acid to palmitoleicacid (16:1) and/or stearic acid to oleic acid (18:1). In the art, Δ15and Δ17 desaturases are also occasionally referred to as “omega-3desaturases”, “w-3 desaturases” and/or “ω-3 desaturases”, based on theirability to convert ω-6 fatty acids into their ω-3 counterparts (e.g.,conversion of LA into ALA and ARA into EPA, respectively). In someembodiments, it may be desirable to empirically determine thespecificity of a particular fatty acid desaturase by transforming asuitable host with the gene for the fatty acid desaturase anddetermining its effect on the fatty acid profile of the host.

For the purposes herein, an enzyme catalyzing the first condensationreaction (i.e., conversion of malonyl-CoA and long-chain acyl-CoA to-ketoacyl-CoA) will be referred to generically as an “elongase”. Ingeneral, the substrate selectivity of elongases is somewhat broad butsegregated by both chain length and the degree of unsaturation.Accordingly, elongases can have different specificities. For example, aC_(14/16) elongase will utilize a C₁₄ substrate (e.g., myristic acid), aC_(16/18) elongase will utilize a C₁₆ substrate (e.g., palmitate), aC_(18/20) elongase (also known as a Δ6 elongase as the terms can be usedinterchangeably) will utilize a C₁₈ substrate (e.g., GLA, STA) and aC_(20/22) elongase will utilize a C₂₀ substrate (e.g., ARA, EPA). Inlike manner, and of particular interest herein, a “Δ9 elongase”catalyzes the conversion of LA to EDA and/or ALA to ETrA. It isimportant to note that some elongases have broad specificity and thus asingle enzyme may be capable of catalyzing several elongase reactions.Thus, for example, a Δ9 elongase may also act as a C_(16/18) elongase,C_(18/20) elongase and/or C_(20/22) elongase and may have alternate, butnot preferred, specificities for Δ5 and Δ6 fatty acids such as EPAand/or GLA, respectively. In preferred embodiments, it may be desirableto empirically determine the specificity of a fatty acid elongase bytransforming a suitable host with the gene for the fatty acid elongaseand determining its effect on the fatty acid profile of the host.Elongase systems generally comprise four enzymes that are responsiblefor elongation of a fatty acid carbon chain to produce a fatty acid thatis two carbons longer than the fatty acid substrate that the elongasesystem acts upon. More specifically, the process of elongation occurs inassociation with fatty acid synthase, whereby CoA is the acyl carrier(Lassner et al., Plant Cell, 8:281-292 (1996)). In the first step, whichhas been found to be both substrate-specific and also rate-limiting,malonyl-CoA is condensed with a long-chain acyl-CoA to yield carbondioxide (CO₂) and a β-ketoacyl-CoA (where the acyl moiety has beenelongated by two carbon atoms). Subsequent reactions include reductionto β-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a second reductionto yield the elongated acyl-CoA. Examples of reactions catalyzed byelongase systems are the conversion of GLA to DGLA, STA to ETA, LA toEDA, ALA to ETrA, ARA to DTA and EPA to DPA.

For the purposes herein, the term “EaD9Elo1” refers to a Δ9 elongaseenzyme (SEQ ID NO:13) isolated from Euglena anabaena, encoded by SEQ IDNO:11 herein. The term “EaD9Elo2” refers to a Δ9 elongase enzyme (SEQ IDNO:14) isolated from E. anabaena, encoded by SEQ ID NO:12 herein.Likewise, the term “EaD9eS” refers to a synthetic Δ9 elongase derivedfrom E. anabaena that is codon-optimized for expression in Yarrowialipolytica (i.e., SEQ ID NOs:26 and 27).

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., an elongase)can convert substrate to product. The conversion efficiency is measuredaccording to the following formula: ([product]/[substrate+product])*100,where ‘product’ includes the immediate product and all products in thepathway derived from it.

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2^(nd) Ed., Plenum, 1980). Within oleaginousmicroorganisms the cellular oil or TAG content generally follows asigmoid curve, wherein the concentration of lipid increases until itreaches a maximum at the late logarithmic or early stationary growthphase and then gradually decreases during the late stationary and deathphases (Yongmanitchai and Ward, Appl. Environ. Microbiol., 57:419-25(1991)). It is not uncommon for oleaginous microorganisms to accumulatein excess of about 25% of their dry cell weight as oil.

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that make oil. Examples of oleaginous yeast include, but are nomeans limited to, the following genera: Yarrowia, Candida, Rhodotorula,Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

The term “Euglenophyceae” refers to a group of unicellular colorless orphotosynthetic flagellates (“euglenoids”) found living in freshwater,marine, soil, and parasitic environments. The class is characterized bysolitary unicells, wherein most are free-swimming and have two flagella(one of which may be nonemergent) arising from an anterior invaginationknown as a reservoir. Photosynthetic euglenoids contain one to manychloroplasts, which vary from minute disks to expanded plates orribbons. Colorless euglenoids depend on osmotrophy or phagotrophy fornutrient assimilation. About 1000 species have been described andclassified into about 40 genera and 6 orders. Examples of Euglenophyceaeinclude, but are no means limited to, the following genera: Euglena,Eutreptiella and Tetruetreptia.

As used herein, “nucleic acid” means a polynucleotide and includessingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Thus, the terms “polynucleotide”, “nucleic acidsequence”, “nucleotide sequence” or “nucleic acid fragment” are usedinterchangeably and refer to a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases. A polynucleotide in the form of a polymer of DNA maybe comprised of one or more segments of cDNA, genomic DNA, syntheticDNA, or mixtures thereof. Nucleotides (usually found in their5′-monophosphate form) are referred to by their single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deosycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridlate, “T” for deosythymidylate,“R” for purines (A or G), “Y” for pyrimidiens (C or T), “K” for G or T,“H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialin the structure, the stability, or the activity of a protein. Becausethey are identified by their high degree of conservation in alignedsequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family.PCT Publications No. WO 2007/061845 and No. WO 2007/061742 describeseven distinct motifs that are associated with Δ9 elongases.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize (under moderately stringent conditions, e.g.,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences disclosed herein and which arefunctionally equivalent to any of the nucleic acid sequences disclosedherein. Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, or 90% sequence identity, upto and including 100% sequence identity (i.e., fully complementary) witheach other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence. Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, theimportant factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth et al., Anal. Biochem., 138:267-284 (1984):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); and, low stringency conditions canutilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point (T_(m)). Using the equation,hybridization and wash compositions, and desired T_(m), those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T_(m) of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Hybridization and/or washconditions can be applied for at least 10, 30, 60, 90, 120 or 240minutes.

“Sequence identity” or “identity” in the context of nucleic acid orpolypeptide sequences refers to the nucleic acid bases or amino acidresidues in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window.

Thus, “percentage of sequence identity” refers to the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the results by 100 to yield the percentage of sequenceidentity. Useful examples of percent sequence identities include, butare not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%,or any integer percentage from 50% to 100%. These identities can bedetermined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters thatoriginally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp, CABIOS, 5:151-153(1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992))and found in the MegAlign™ program of the LASERGENE bioinformaticscomputing suite (supra). For multiple alignments, the default valuescorrespond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using the Clustal V method are KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids theseparameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.After alignment of the sequences using the Clustal V program, it ispossible to obtain a “percent identity” by viewing the “sequencedistances” table in the same program.

“BLASTN method of alignment” is an algorithm provided by the NationalCenter for Biotechnology Information (NCBI) to compare nucleotidesequences using default parameters.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or anyinteger percentage from 50% to 100%. Indeed, any integer amino acididentity from 50% to 100% may be useful in describing the presentinvention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, ofinterest is any full-length or partial complement of this isolatednucleotide fragment.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the instant euglenoidpolypeptides as set forth in SEQ ID NO:13 and SEQ ID NO:14. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. Accordingly, the genes can be tailored for optimal gene expressionbased on optimization of nucleotide sequence to reflect the codon biasof the host cell. The skilled artisan appreciates the likelihood ofsuccessful gene expression if codon usage is biased towards those codonsfavored by the host. Determination of preferred codons can be based on asurvey of genes derived from the host cell, where sequence informationis available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and that may refer to the coding region alone or may includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. A“codon-optimized gene” is a gene having its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to: promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters thatcause a gene to be expressed at almost all stages of development, arecommonly referred to as “constitutive promoters”. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences, especially at its 5′ end, have not been completely defined,DNA fragments of some variation may have identical promoter activity.

A promoter sequence may consist of proximal and more distal upstreamelements, the latter elements often referred to as enhancers and/orsilencers. Accordingly, an “enhancer” is a DNA sequence that canstimulate promoter activity, and may be an innate element of thepromoter or a heterologous element inserted to enhance the level orstage-specific activity of a promoter. A “silencer” is a DNA sequencethat can repress promoter activity, and may be an innate element of thepromoter or a heterologous element inserted to repress the level orstage-specific activity of a promoter.

“Translation leader sequence” refers to a polynucleotide sequencelocated between the transcription start site of a gene and the codingsequence. The translation leader sequence is present in the fullyprocessed mRNA upstream of the translation start sequence. Thetranslation leader sequence may affect processing of the primarytranscript to mRNA, mRNA stability or translation efficiency. Examplesof translation leader sequences have been described (Turner, R. andFoster, G. D., Mol. Biotechnol., 3:225-236 (1995)).

The terms “3′ non-coding sequences”, “transcription terminator” and“termination sequences” refer to DNA sequences located downstream of acoding sequence. This includes polyadenylation recognition sequences andother sequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The 3′ region can influence thetranscription, RNA processing or stability, or translation of theassociated coding sequence.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript. A RNA transcript is referred toas the mature RNA when it is a RNA sequence derived frompost-transcriptional processing of the primary transcript. “MessengerRNA” or “mRNA” refers to the RNA that is without introns and that can betranslated into protein by the cell. “cDNA” refers to a DNA that iscomplementary to, and synthesized from, a mRNA template using the enzymereverse transcriptase. The cDNA can be single-stranded or converted intodouble-stranded form using the Klenow fragment of DNA polymerase I.“Sense” RNA refers to RNA transcript that includes the mRNA and can betranslated into protein within a cell or in vitro. “Antisense RNA”refers to an RNA transcript that is complementary to all or part of atarget primary transcript or mRNA, and that blocks the expression of atarget gene (U.S. Pat. No. 5,107,065). The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to antisense RNA, ribozymeRNA, or other RNA that may not be translated but yet has an effect oncellular processes. The terms “complement” and “reverse complement” areused interchangeably herein with respect to mRNA transcripts, and aremeant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragments of the invention. Expression may also refer totranslation of mRNA into a protein (either precursor or mature).

“Mature” protein refers to a post-translationally processed polypeptide(i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed). “Precursor” protein refers tothe primary product of translation of mRNA (i.e., with pre- andpropeptides still present). Pre- and propeptides may be but are notlimited to intracellular localization signals.

The terms “plasmid” and “vector” refer to an extra chromosomal elementoften carrying genes that are not part of the central metabolism of thecell, and usually in the form of circular double-stranded DNA fragments.Such elements may be autonomously replicating sequences, genomeintegrating sequences, phage or nucleotide sequences, linear orcircular, of a single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction which is capable of introducing anexpression cassette(s) into a cell.

The term “expression cassette” refers to a fragment of DNA comprisingthe coding sequence of a selected gene and regulatory sequencespreceding (5′ non-coding sequences) and following (3′ non-codingsequences) the coding sequence that are required for expression of theselected gene product. Thus, an expression cassette is typicallycomposed of: (1) a promoter sequence; (2) a coding sequence (i.e., ORF);and, (3) a 3′ untranslated region (i.e., a terminator) that, ineukaryotes, usually contains a polyadenylation site. The expressioncassette(s) is usually included within a vector, to facilitate cloningand transformation. Different expression cassettes can be transformedinto different organisms including bacteria, yeast, plants and mammaliancells, as long as the correct regulatory sequences are used for eachhost.

A “recombinant DNA construct” (also referred to interchangeably hereinas a “expression construct” and “construct”) comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such aconstruct may be used by itself or may be used in conjunction with avector. If a vector is used, then the choice of vector is dependent uponthe method that will be used to transform host cells as is well known tothose skilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the invention. The skilled artisan will also recognize thatdifferent independent transformation events will result in differentlevels and patterns of expression (Jones et al., EMBO J., 4:2411-2418(1985); De Almeida et al., Mol. Gen. Genetics, 218:78-86 (1989)), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others.

The term “introduced” means providing a nucleic acid (e.g., expressioncassette) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct or expression cassette) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

As used herein, “transgenic” refers to a cell which comprises within itsgenome a heterologous polynucleotide. Preferably, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of anexpression cassette. Transgenic is used herein to include any cell orcell line, the genotype of which has been altered by the presence ofheterologous nucleic acids including those transgenics initially soaltered as well as those created by mating from the initial transgenicwith different mating types. The term “transgenic” as used herein doesnot encompass the alteration of the genome (chromosomal orextra-chromosomal) by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989); bySilhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with GeneFusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984);and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken,N.J. (1987). Transformation methods are well known to those skilled inthe art and are described infra. An Overview: Microbial Biosynthesis OfFatty Acids And Triacylglycerols

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium. This process, leading to the de novo synthesis of freepalmitate (16:0) in oleaginous microorganisms, is described in detail inU.S. Pat. No. 7,238,482. Palmitate is the precursor of longer-chainsaturated and unsaturated fatty acid derivates, which are formed throughthe action of elongases and desaturases (FIG. 1).

TAGs (the primary storage unit for fatty acids) are formed by a seriesof reactions that involve: (1) the esterification of one molecule ofacyl-CoA to glycerol-3-phosphate via an acyltransferase to producelysophosphatidic acid; (2) the esterification of a second molecule ofacyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate(commonly identified as phosphatidic acid); (3) removal of a phosphateby phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and,(4) the addition of a third fatty acid by the action of anacyltransferase to form TAG. A wide spectrum of fatty acids can beincorporated into TAGs, including saturated and unsaturated fatty acidsand short-chain and long-chain fatty acids.

Biosynthesis of Omega Fatty Acids

The metabolic process wherein oleic acid is converted to ω-3/ω-6 fattyacids involves elongation of the carbon chain through the addition ofcarbon atoms and desaturation of the molecule through the addition ofdouble bonds. This requires a series of special desaturation andelongation enzymes present in the endoplasmic reticulim membrane.However, as seen in FIG. 1 and as described below, there are oftenmultiple alternate pathways for production of a specific ω-3/ω-6 fattyacid.

Specifically, all pathways require the initial conversion of oleic acidto LA, the first of the ω-6 fatty acids, by a Δ12 desaturase. Then,using the “Δ9 elongase/Δ8 desaturase pathway” and LA as substrate, longchain ω-6 fatty acids are formed as follows: (1) LA is converted to EDAby a Δ9 elongase; (2) EDA is converted to DGLA by a Δ8 desaturase; (3)DGLA is converted to ARA by a Δ5 desaturase; (4) ARA is converted to DTAby a C_(20/22) elongase; and, (5) DTA is converted to DPAn-6 by a Δ4desaturase. Alternatively, the “Δ9 elongase/Δ8 desaturase pathway” canuse ALA as substrate to produce long chain ω-3 fatty acids as follows:(1) LA is converted to ALA, the first of the ω-3 fatty acids, by a Δ15desaturase; (2) ALA is converted to ETrA by a Δ9 elongase; (3) ETrA isconverted to ETA by a Δ8 desaturase; (4) ETA is converted to EPA by a Δ5desaturase; (5) EPA is converted to DPA by a C_(20/22) elongase; and,(6) DPA is converted to DHA by a Δ4 desaturase. Optionally, ω-6 fattyacids may be converted to ω-3 fatty acids; for example, ALA is producedfrom LA by Δ15 desaturase activity; ETA and EPA are produced from DGLAand ARA, respectively, by Δ17 desaturase activity.

Alternate pathways for the biosynthesis of ω-3/ω-6 fatty acids utilize aΔ6 desaturase and C_(18/20) elongase (i.e., the “Δ6 desaturase/Δ6elongase pathway”). More specifically, LA and ALA may be converted toGLA and STA, respectively, by a Δ6 desaturase; then, a C_(18/20)elongase converts GLA to DGLA and/or STA to ETA. Downstream PUFAs aresubsequently formed as described above.

It is contemplated that the particular functionalities required to beintroduced into a specific host organism for production of ω-3/ω-6 fattyacids will depend on the host cell (and its native PUFA profile and/ordesaturase/elongase profile), the availability of substrate, and thedesired end product(s). For example, expression of the Δ9 elongase/Δ8desaturase pathway may be preferred in some embodiments, as opposed toexpression of the Δ6 desaturase/Δ6 elongase pathway, since PUFAsproduced via the former pathway are devoid of GLA and/or STA.

One skilled in the art will be able to identify various candidate genesencoding each of the enzymes desired for ω-3/ω-6 fatty acidbiosynthesis. Useful desaturase and elongase sequences may be derivedfrom any source, e.g., isolated from a natural source (from bacteria,algae, fungi, plants, animals, etc.), produced via a semi-syntheticroute or synthesized de novo. Although the particular source of thedesaturase and elongase genes introduced into the host is not critical,considerations for choosing a specific polypeptide having desaturase orelongase activity include: (1) the substrate specificity of thepolypeptide; (2) whether the polypeptide or a component thereof is arate-limiting enzyme; (3) whether the desaturase or elongase isessential for synthesis of a desired PUFA; (4) co-factors required bythe polypeptide; and/or, (5) whether the polypeptide was modified afterits production (e.g., by a kinase or a prenyltransferase). The expressedpolypeptide preferably has parameters compatible with the biochemicalenvironment of its location in the host cell (see U.S. Pat. No.7,238,482 for additional details).

In additional embodiments, it will also be useful to consider theconversion efficiency of each particular desaturase and/or elongase.More specifically, since each enzyme rarely functions with 100%efficiency to convert substrate to product, the final lipid profile ofunpurified oils produced in a host cell will typically be a mixture ofvarious PUFAs consisting of the desired ω-3/ω-6 fatty acid, as well asvarious upstream intermediary PUFAs. Thus, each enzyme's conversionefficiency is also a variable to consider, when optimizing biosynthesisof a desired fatty acid.

With each of the considerations above in mind, candidate genes havingthe appropriate desaturase and elongase activities (e.g., Δ6desaturases, C_(18/20) elongases, Δ5 desaturases, Δ17 desaturases, Δ15desaturases, Δ9 desaturases, Δ12 desaturases, C_(14/16) elongases,C_(16/18) elongases, Δ9 elongases, Δ8 desaturases, Δ4 desaturases andC_(20/22) elongases) can be identified according to publicly availableliterature (e.g., GenBank), the patent literature, and experimentalanalysis of organisms having the ability to produce PUFAs. These geneswill be suitable for introduction into a specific host organism, toenable or enhance the organism's synthesis of PUFAs.

Sequence Identification of Novel Δ9 Elongases

In the present invention, nucleotide sequences encoding Δ9 elongaseshave been isolated from Euglena anabaena, as summarized below in Table3.

TABLE 3 Summary Of Euglena anabaena Δ9 Elongases Nucleotide Amino AcidAbbreviation SEQ ID NO SEQ ID NO EaD9Elo1 11 13 EaD9Elo2  12 14 EaD9eS26 27 *Note:  SEQ ID NO: 27 is identical in sequence to SEQ ID NO: 13 

Thus, the present invention concerns an isolated polynucleotidecomprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having Δ9        elongase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:13 or SEQ ID NO:14;    -   (b) a nucleotide sequence encoding a polypeptide having Δ9        elongase activity, wherein the nucleotide sequence has at least        80% sequence identity, based on the BLASTN method of alignment,        when compared to a nucleotide sequence as set forth in SEQ ID        NO:11, SEQ ID NO:12 or SEQ ID NO:26; or,    -   (c) a complement of the nucleotide sequence of (a) or (b),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

In still another aspect, this invention concerns an isolatedpolynucleotide comprising a nucleotide sequence encoding a polypeptidehaving Δ9 elongase activity, wherein the nucleotide sequence has atleast 90% sequence identity, based on the BLASTN method of alignment,when compared to a nucleotide sequence as set forth in SEQ ID NO:11, SEQID NO:12 or SEQ ID NO:26.

More preferred amino acid fragments that are at least about 80%-90%identical are particularly suitable and those sequences that are atleast about 90%-95% identical are most preferred. Similarly, preferredΔ9 elongase encoding nucleic acid sequences corresponding to the instantORFs are those encoding active proteins and which are at least about80%-90% identical; those sequences that are at least about 90%-95%identical are most preferred.

In alternate embodiments, the instant EaD9Elo1 and/or EaD9Elo2 sequencescan be codon-optimized for expression in a particular host organism. Asis well known in the art, this can be a useful means to further optimizethe expression of the enzyme in the alternate host, since use ofhost-preferred codons can substantially enhance the expression of theforeign gene encoding the polypeptide. In general, host-preferred codonscan be determined within a particular host species of interest byexamining codon usage in proteins (preferably those expressed in thelargest amount) and determining which codons are used with highestfrequency. Then, the coding sequence for a polypeptide of interesthaving e.g., elongase activity can be synthesized in whole or in partusing the codons preferred in the host species. All (or portions) of theDNA also can be synthesized to remove any destabilizing sequences orregions of secondary structure that would be present in the transcribedmRNA. All (or portions) of the DNA also can be synthesized to alter thebase composition to one more preferable in the desired host cell.

In one embodiment of the invention herein, EaD9Elo1 (SEQ ID NO:11) wascodon-optimized for expression in Yarrowia lipolytica. This was possiblebased on previous determination of the Y. lipolytica codon usageprofile, identification of those codons that were preferred, anddetermination of the consensus sequence around the ‘ATG’ initiationcodon (see U.S. Pat. No. 7,238,482 and U.S. Pat. No. 7,125,672). Theresultant synthetic gene is referred to as EaD9ES (SEQ ID NO:26). Theprotein sequence encoded by the codon-optimized Δ9 elongase gene (i.e.,SEQ ID NO:27) is identical to that of the wildtype protein sequence(i.e., SEQ ID NO:13). Similar techniques could be utilized to produce asynthetic Δ9 elongase derived from EaD9Elo2 (SEQ ID NO:12) forexpression in Y. lipolytica.

One skilled in the art would be able to use the teachings herein tocreate various other codon-optimized Δ9 elongase proteins suitable foroptimal expression in alternate hosts (i.e., other than Yarrowialipolytica), based on the wildtype EaD9Elo1 and/or EaD9Elo2 sequences.Accordingly, the instant invention relates to any codon-optimized Δ9elongase protein that is derived from the wildtype nucleotide sequencesof EaD9Elo1 (SEQ ID NO:11) or EaD9Elo2 (SEQ ID NO:12). This includes,but is not limited to, the nucleotide sequence set forth in SEQ IDNO:26, which encodes a synthetic Δ9 elongase protein (i.e., EaD9eS) thatwas codon-optimized for expression in Yarrowia lipolytica. In alternateembodiments, it may be desirable to modify a portion of the codonsencoding EaD9Elo1 and/or EaD9Elo2 to enhance expression of the gene in ahost organism including, but not limited to, a plant or plant part,algae, bacteria, alternate yeast, euglenoid, stramenopiles or fungi.

Identification and Isolation of Homologs

Any of the instant elongase sequences (i.e., EaD9Elo1, EaD9Elo2, orEaD9eS) or portions thereof may be used to search for Δ9 elongasehomologs in the same or other bacterial, algal, fungal, euglenoid orplant species using sequence analysis software. In general, suchcomputer software matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.

Alternatively, any of the instant elongase sequences or portions thereofmay also be employed as hybridization reagents for the identification ofΔ9 elongase homologs. The basic components of a nucleic acidhybridization test include a probe, a sample suspected of containing thegene or gene fragment of interest and a specific hybridization method.Probes of the present invention are typically single-stranded nucleicacid sequences that are complementary to the nucleic acid sequences tobe detected. Probes are “hybridizable” to the nucleic acid sequence tobe detected. Although the probe length can vary from 5 bases to tens ofthousands of bases, typically a probe length of about 15 bases to about30 bases is suitable. Only part of the probe molecule need becomplementary to the nucleic acid sequence to be detected. In addition,the complementarity between the probe and the target sequence need notbe perfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added (e.g., guanidinium chloride, guanidiniumthiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodiumperchlorate, rubidium tetrachloroacetate, potassium iodide, cesiumtrifluoroacetate). If desired, one can add formamide to thehybridization mixture, typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

In additional embodiments, any of the Δ9 elongase nucleic acid fragmentsdescribed herein (or any homologs identified thereof) may be used toisolate genes encoding homologous proteins from the same or otherbacterial, algal, fungal, euglenoid or plant species. Isolation ofhomologous genes using sequence-dependent protocols is well known in theart. Examples of sequence-dependent protocols include, but are notlimited to: (1) methods of nucleic acid hybridization; (2) methods ofDNA and RNA amplification, as exemplified by various uses of nucleicacid amplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor et al., Proc. Acad. Sci. U.S.A., 82:1074 (1985); or stranddisplacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and, (3) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to the Δ9elongases described herein could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from e.g., any desired yeast or fungus usingmethodology well known to those skilled in the art (wherein thoseorganisms producing EDA and/or ETrA would be preferred). Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis,supra). Moreover, the entire sequences can be used directly tosynthesize DNA probes by methods known to the skilled artisan (e.g.,random primers DNA labeling, nick translation or end-labelingtechniques), or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of (or full-length of) the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length DNA fragments under conditions ofappropriate stringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, V A; and Rychlik, W., In Methods in MolecularBiology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used in PCRprotocols to amplify longer nucleic acid fragments encoding homologousgenes from DNA or RNA. PCR may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding eukaryotic genes.

Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., Proc. Acad. Sci. U.S.A.,85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of theregion between a single point in the transcript and the 3′ or 5′ end.Primers oriented in the 3′ and 5′ directions can be designed from theinstant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (Gibco/BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragmentscan be isolated (Ohara et al., Proc. Acad. Sci. U.S.A., 86:5673 (1989);Loh et al., Science, 243:217 (1989)).

In other embodiments, any of the Δ9 elongase nucleic acid fragmentsdescribed herein (or any homologs identified thereof) may be used forcreation of new and/or improved fatty acid elongases. As is well knownin the art, in vitro mutagenesis and selection, chemical mutagenesis,“gene shuffling” methods or other means can be employed to obtainmutations of naturally occurring elongase genes (wherein such mutationsmay include deletions, insertions and point mutations, or combinationsthereof). This would permit production of a polypeptide having elongaseactivity, respectively, in vivo with more desirable physical and kineticparameters for function in the host cell such as a longer half-life or ahigher rate of production of a desired PUFA. Or, if desired, the regionsof a polypeptide of interest (i.e., a Δ9 elongase) important forenzymatic activity can be determined through routine mutagenesis,expression of the resulting mutant polypeptides and determination oftheir activities. An overview of these techniques is described in U.S.Pat. No. 7,238,482. All such mutant proteins and nucleotide sequencesencoding them that are derived from EaD9Elo1, EaD9Elo2 and EaD9eS arewithin the scope of the present invention.

Alternatively, improved fatty acids may be synthesized by domainswapping, wherein a functional domain from any of the Δ9 elongasenucleic acid fragments described herein are exchanged with a functionaldomain in an alternate elongase gene to thereby result in a novelprotein. As used herein, “domain” or “functional domain” refer tonucleic acid sequence(s) that are capable of eliciting a biologicalresponse in microbes.

Methods for Production of Various Omega-3 and/or Omega-6 Fatty Acids

It is expected that introduction of chimeric genes encoding the Δ9elongases described herein (i.e., EaD9Elo1, EaD9Elo2, EaD9eS or othermutant enzymes, codon-optimized enzymes or homologs thereof), under thecontrol of the appropriate promoters will result in increased productionof EDA and/or ETrA in the transformed host organism, respectively. Assuch, the present invention encompasses a method for the directproduction of PUFAs comprising exposing a fatty acid substrate (i.e., LAand/or ALA) to the elongase enzymes described herein (e.g., EaD9Elo1,EaD9Elo2 or EaD9eS), such that the substrate is converted to the desiredfatty acid product (i.e., EDA and/or ETrA, respectively).

More specifically, it is an object of the present invention to provide amethod for the production of EDA in a microbial host cell (e.g., yeast,algae, bacteria, euglenoids, stramenopiles and fungi), wherein themicrobial host cell comprises:

-   -   (a) a recombinant nucleotide molecule encoding a Δ9 elongase        polypeptide having at least 80% amino acid identity, based on        the Clustal V method of alignment, when compared to a        polypeptide having an amino acid sequence as set forth in SEQ ID        NO:13 or SEQ ID NO:14; and,    -   (b) a source of LA;        wherein the microbial host cell is grown under conditions such        that the nucleic acid fragment encoding the Δ9 elongase is        expressed and the LA is converted to EDA, and wherein the EDA is        optionally recovered.

In alternate embodiments of the present invention, the Δ9 elongase maybe used for the conversion of ALA to ETrA. Accordingly the inventionprovides a method for the production of ETrA, wherein the microbial hostcell comprises:

-   -   (a) a recombinant nucleotide molecule encoding a Δ9 elongase        polypeptide having at least 80% amino acid identity, based on        the Clustal V method of alignment, when compared to a        polypeptide having an amino acid sequence as set forth in SEQ ID        NO:13 or SEQ ID NO:14; and,    -   (b) a source of ALA;        wherein the microbial host cell is grown under conditions such        that the nucleic acid fragment encoding the Δ9 elongase is        expressed and the ALA is converted to ETrA, and wherein the ETrA        is optionally recovered.

Alternatively, each Δ9 elongase gene and its corresponding enzymeproduct described herein can be used indirectly for the production ofvarious ω-6 and ω-3 PUFAs (see FIG. 1 and U.S. Pat. No. 7,238,482).Indirect production of ω-3/ω-6 PUFAs occurs wherein the fatty acidsubstrate is converted indirectly into the desired fatty acid product,via means of an intermediate step(s) or pathway intermediate(s). Thus,it is contemplated that the Δ9 elongases described herein (i.e.,EaD9Elo1, EaD9Elo2, EaD9eS or other mutant enzymes, codon-optimizedenzymes or homologs thereof) may be expressed in conjunction withadditional genes encoding enzymes of the PUFA biosynthetic pathway(e.g., Δ6 desaturases, C_(18/20) elongases, Δ17 desaturases, Δ8desaturases, Δ15 desaturases, Δ9 desaturases, Δ12 desaturases, C_(14/16)elongases, C_(16/18) elongases, Δ9 elongases, Δ5 desaturases, Δ4desaturases, C_(20/22) elongases) to result in higher levels ofproduction of longer-chain ω-3/ω-6 fatty acids (e.g., ARA, EPA, DTA,DPAn-6, DPA and/or DHA).

In preferred embodiments, the Δ9 elongases of the present invention willminimally be expressed in conjunction with a Δ8 desaturase (e.g., fromEuglena gracilis [Wallis et al., Arch. Biochem. and Biophys.,365(2):307-316 (May 1999); PCT Publication No. WO 2000/34439; U.S. Pat.No. 6,825,017; PCT Publication No. WO 2004/057001; PCT Publication No.WO 2006/012325; U.S. Pat. No. 7,256,033; U.S. patent application Ser.No. 11/635,258]; from Acanthamoeba castellanii [Sayanova et al., FEBSLett., 580:1946-1952 (2006)]; from Pavlova salina [PCT Publication No.WO 2005/103253]; from Pavlova lutheri [PCT Publication No. WO2007/127381]; from Tetruetreptia pomquetensis CCMP1491 [U.S. patentapplication Ser. No. 11/876,115]; from Eutreptiella sp. CCMP389 [U.S.patent application Ser. No. 11/876,115]; from Eutreptiella cf _(—)gymnastica CCMP1594 [U.S. patent application Ser. No. 11/876,115; and,from Euglena anabaena [described in co-pending U.S. patent applicationSer. Nos. 12/099,799 and No. 12/099,811]). However, the particular genesincluded within a particular expression cassette will depend on the hostcell (and its PUFA profile and/or desaturase/elongase profile), theavailability of substrate and the desired end product(s).

In alternative embodiments, it may be useful to disrupt a hostorganism's native Δ9 elongase, based on the complete sequences describedherein, the complement of those complete sequences, substantial portionsof those sequences, codon-optimized elongases derived therefrom andthose sequences that are substantially homologous thereto.

Microbial Expression Systems, Cassettes and Vectors

The Δ9 elongase genes and gene products described herein (i.e.,EaD9Elo1, EaD9Elo2, EaD9eS or other mutant enzymes, codon-optimizedenzymes or homologs thereof) may be expressed in heterologous microbialhost cells, particularly in the cells of oleaginous yeasts (e.g.,Yarrowia lipolytica).

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of any of the geneproducts of the instant sequences. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the encoded enzymes.

Vectors (e.g., constructs, plasmids) and DNA expression cassettes usefulfor the transformation of suitable microbial host cells are well knownin the art. The specific choice of sequences present in the construct isdependent upon the desired expression products (supra), the nature ofthe host cell and the proposed means of separating transformed cellsversus non-transformed cells. Typically, however, the vector contains atleast one expression cassette, a selectable marker and sequencesallowing autonomous replication or chromosomal integration. Suitableexpression cassettes comprise a region 5′ of the gene that controlstranscription (e.g., a promoter), the gene coding sequence, and a region3′ of the DNA fragment that controls transcriptional termination (i.e.,a terminator). It is most preferred when both control regions arederived from genes from the transformed microbial host cell, although itis to be understood that such control regions need not be derived fromthe genes native to the specific species chosen as a production host.

Transcriptional control regions (also initiation control regions orpromoters) which are useful to drive expression of the instant Δ9elongase ORFs in the desired microbial host cell are numerous andfamiliar to those skilled in the art. Virtually any promoter (i.e.,native, synthetic, or chimeric) capable of directing expression of thesegenes in the selected host cell is suitable for the present invention,although transcriptional and translational regions from the host speciesare particularly useful. Expression in a microbial host cell can beaccomplished in an induced or constitutive fashion. Induced expressioncan be accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest, while constitutive expressioncan be achieved by the use of a constitutive promoter operably linked tothe gene of interest. As an example, when the host cell is yeast,transcriptional and translational regions functional in yeast cells areprovided, particularly from the host species (e.g., see PatentPublication No. US-2006-0115881-A1 for preferred transcriptionalinitiation regulatory regions for use in Yarrowia lipolytica). Any oneof a number of regulatory sequences can be used, depending upon whetherconstitutive or induced transcription is desired, the efficiency of thepromoter in expressing the ORF of interest, the ease of construction andthe like.

Nucleotide sequences surrounding the translational initiation codon‘ATG’ have been found to affect expression in yeast cells. If thedesired polypeptide is poorly expressed in yeast, the nucleotidesequences of exogenous genes can be modified to include an efficientyeast translation initiation sequence to obtain optimal gene expression.For expression in yeast, this can be done by site-directed mutagenesisof an inefficiently expressed gene by fusing it in-frame to anendogenous yeast gene, preferably a highly expressed gene.Alternatively, one can determine the consensus translation initiationsequence in the host and engineer this sequence into heterologous genesfor their optimal expression in the host of interest.

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same anddifferent genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Termination controlregions may also be derived from various genes native to the preferredhosts. In alternate embodiments, the 3′-region can also be synthetic, asone of skill in the art can utilize available information to design andsynthesize a 3′-region sequence that functions as a transcriptionterminator. Optionally, a termination site may be unnecessary; however,it is most preferred if included.

As one of skill in the art is aware, merely inserting a gene into acloning vector does not ensure that it will be successfully expressed atthe level needed. In response to the need for a high expression rate,many specialized expression vectors have been created by manipulating anumber of different genetic elements that control aspects oftranscription, translation, protein stability, oxygen limitation andsecretion from the microbial host cell. More specifically, some of themolecular features that have been manipulated to control gene expressioninclude: the nature of the relevant transcriptional promoter andterminator sequences; the number of copies of the cloned gene (whereinadditional copies may be cloned within a single expression constructand/or additional copies may be introduced into the host cell byincreasing the plasmid copy number or by multiple integration of thecloned gene into the genome); whether the gene is plasmid-borne orintegrated into the genome of the host cell; the final cellular locationof the synthesized foreign protein; the efficiency of translation andcorrect folding of the protein in the host organism; the intrinsicstability of the mRNA and protein of the cloned gene within the hostcell; and, the codon usage within the cloned gene, such that itsfrequency approaches the frequency of preferred codon usage of the hostcell. Each of these types of modifications are encompassed in thepresent invention, as means to further optimize expression of the Δ9elongase described herein.

Transformation of Microbial Host Cells

Once a DNA cassette that is suitable for expression in an appropriatemicrobial host cell has been obtained (e.g., a chimeric gene comprisinga promoter, ORF and terminator), it is placed in a plasmid vectorcapable of autonomous replication in a host cell, or it is directlyintegrated into the genome of the host cell. Integration of expressioncassettes can occur randomly within the host genome or can be targetedthrough the use of constructs containing regions of homology with thehost genome sufficient to target recombination within the host locus.Where constructs are targeted to an endogenous locus, all or some of thetranscriptional and translational regulatory regions can be provided bythe endogenous locus.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other construct(s) to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct(s) can be experimentallydetermined so that all introduced genes are expressed at the necessarylevels to provide for synthesis of the desired products.

Constructs comprising the gene(s) of interest may be introduced into amicrobial host cell by any standard technique. These techniques includetransformation (e.g., lithium acetate transformation [Methods inEnzymology, 194:186-187 (1991)]), protoplast transformation, bolisticimpact, electroporation, microinjection, or any other method thatintroduces the gene(s) of interest into the host cell.

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed”, “transformant” or “recombinant” herein. Thus, theterm “transformed” and “recombinant” are used interchangeably herein.The transformed host will have at least one copy of the expressionconstruct and may have two or more, depending upon whether theexpression cassette is integrated into the genome or is present on anextrachromosomal element having multiple copy numbers.

The transformed host cell can be identified by various selectiontechniques, as described in U.S. Pat. No. 7,238,482, U.S. Pat. No.7,259,255 and PCT Publication No. WO 2006/052870.

Following transformation, substrates suitable for the instant Δ9elongase (and, optionally other PUFA enzymes that are co-expressedwithin the host cell) may be produced by the host either naturally ortransgenically, or they may be provided exogenously.

Preferred Microbial Hosts for Recombinant Expression

Microbial host cells for expression of the instant genes and nucleicacid fragments may include hosts that grow on a variety of feedstocks,including simple or complex carbohydrates, fatty acids, organic acids,oils, glycerol and alcohols, and/or hydrocarbons over a wide range oftemperature and pH values. Based on the needs of the Applicants'Assignee, the genes described in the instant invention have beenexpressed in an oleaginous yeast (and in particular Yarrowialipolytica); however, it is contemplated that because transcription,translation and the protein biosynthetic apparatus are highly conserved,any bacteria, yeast, algae, euglenoid, stramenopiles and/or fungus willbe a suitable microbial host for expression of the present nucleic acidfragments.

Preferred microbial hosts, however, are oleaginous organisms, such asoleaginous yeasts. These organisms are naturally capable of oilsynthesis and accumulation, wherein the oil can comprise greater thanabout 25% of the cellular dry weight, more preferably greater than about30% of the cellular dry weight, and most preferably greater than about40% of the cellular dry weight. Genera typically identified asoleaginous yeast include, but are not limited to: Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.More specifically, illustrative oil-synthesizing yeasts include:Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candidarevkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporonpullans, T. cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowialipolytica (formerly classified as Candida lipolytica). In alternateembodiments, oil biosynthesis may be genetically engineered such thatthe microbial host cell (e.g., a yeast) can produce more than 25% oil ofthe cellular dry weight, and thereby be considered oleaginous.

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in afurther embodiment, most preferred are the Y. lipolytica strainsdesignated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/orLGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol.,82(1):43-9 (2002)).

Historically, various strains of Y. lipolytica have been used for themanufacture and production of: isocitrate lyase; lipases;polyhydroxyalkanoates; citric acid; erythritol; 2-oxoglutaric acid;γ-decalactone; γ-dodecalatone; and pyruvic acid. Specific teachingsapplicable for transformation of oleaginous yeasts (i.e., Yarrowialipolytica) include U.S. Pat. No. 4,880,741 and U.S. Pat. No. 5,071,764and Chen, D. C. et al. (Appl. Microbiol. Biotechnol., 48(2):232-235(1997)). Specific teachings applicable for engineering ARA, EPA and DHAproduction in Y. lipolytica are provided in U.S. patent application Ser.No. 11/264,784, U.S. patent application Ser. No. 11/265,761, and U.S.patent application Ser. No. 11/264,737, respectively. The preferredmethod of expressing genes in this yeast is by integration of linear DNAinto the genome of the host; and, integration into multiple locationswithin the genome can be particularly useful when high level expressionof genes are desired [e.g., in the Ura3 locus (GenBank Accession No.AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), theLys5 gene locus (GenBank Accession No. M34929), the Aco2 gene locus(GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3: GenBankAccession No. XP_(—)503244; or, Aco3: GenBank Accession No. AJ001301),the Δ12 desaturase gene locus (U.S. Pat. No. 7,214,491), the Lip1 genelocus (GenBank Accession No. Z50020), the Lip2 gene locus (GenBankAccession No. AJ012632), the SCP2 gene locus (GenBank Accession No.AJ431362), and/or the Pex10 gene locus (GenBank Accession No.CAG81606)].

Preferred selection methods for use in Yarrowia lipolytica areresistance to kanamycin, hygromycin and the amino glycoside G418, aswell as ability to grow on media lacking uracil, leucine, lysine,tryptophan or histidine. In alternate embodiments, 5-fluoroorotic acid(5-fluorouracil-6-carboxylic acid monohydrate; “5-FOA”) is used forselection of yeast Ura⁻ mutants. The compound is toxic to yeast cellsthat possess a functioning URA3 gene encoding orotidine 5′-monophosphatedecarboxylase (OMP decarboxylase); thus, based on this toxicity, 5-FOAis especially useful for the selection and identification of Ura⁻ mutantyeast strains (Bartel, P. L. and Fields, S., Yeast 2-Hybrid System,Oxford University: New York, v. 7, pp 109-147, 1997; see also PCTPublication No. WO 2006/052870 for 5-FOA use in Yarrowia).

An alternate preferred selection method for use in Yarrowia relies on adominant, non-antibiotic marker for Yarrowia lipolytica based onsulfonylurea (chlorimuron ethyl; E. I. duPont de Nemours & Co., Inc.,Wilmington, Del.) resistance. More specifically, the marker gene is anative acetohydroxyacid synthase (AHAS or acetolactate synthase; E.C.4.1.3.18) that has a single amino acid change (W497L) that conferssulfonyl urea herbicide resistance (PCT Publication No. WO 2006/052870).AHAS is the first common enzyme in the pathway for the biosynthesis ofbranched-chain amino acids (i.e., valine, leucine, isoleucine) and it isthe target of the sulfonylurea and imidazolinone herbicides.

Other preferred microbial hosts include oleaginous bacteria, algae,euglenoids, stramenopiles and other fungi; and, within this broad groupof microbial hosts, of particular interest are microorganisms thatsynthesize ω-3/ω-6 fatty acids (or those that can be geneticallyengineered for this purpose [e.g., other yeast such as Saccharomycescerevisiae]). Thus, for example, transformation of Mortierella alpina(which is commercially used for production of ARA) with any of thepresent Δ9 elongase genes under the control of inducible or regulatedpromoters could yield a transformant organism capable of synthesizingincreased quantities of EDA; this could be converted to increasedquantities of DGLA if a Δ8 desaturase gene was co-expressed. The methodof transformation of M. alpina is described by Mackenzie et al. (Appl.Environ. Microbiol., 66:4655 (2000)). Similarly, methods fortransformation of Thraustochytriales microorganisms (e.g.,Thraustochytrium, Schizochytrium) are disclosed in U.S. Pat. No.7,001,772.

Irrespective of the host selected for expression of the Δ9 elongasesdescribed herein, it may be necessary to screen multiple transformantsto obtain a strain displaying the desired expression level and pattern.Such screening may be accomplished by Southern analysis of DNA blots(Southern, J. Mol. Biol., 98:503 (1975)), Northern analysis of mRNAexpression (Kroczek, J. Chromatogr. Biomed. Appl., 618(1-2):133-145(1993)), Western and/or Elisa analyses of protein expression, phenotypicanalysis or GC analysis of the PUFA products.

Based on the teachings described above, in one embodiment this inventionis drawn to a method of producing either EDA or ETrA, respectively,comprising:

-   -   (a) providing an oleaginous yeast (e.g., Yarrowia lipolytica)        comprising:        -   (i) a first recombinant nucleotide molecule encoding a Δ9            elongase polypeptide, operably linked to at least one            regulatory sequence; and,        -   (ii) a source of elongase substrate consisting of LA and/or            ALA, respectively; and,    -   (b) growing the yeast of step (a) in the presence of a suitable        fermentable carbon source wherein the gene encoding the Δ9        elongase polypeptide is expressed and LA is converted to EDA        and/or ALA is converted to ETrA, respectively; and,    -   (c) optionally recovering the EDA and/or ETrA, respectively, of        step (b).        Substrate feeding may be required.

The nucleotide sequence of the gene encoding a Δ9 elongase may beselected from the group consisting of SEQ ID NO:11 and SEQ ID NO:12. Inalternate embodiments, the nucleotide sequence of the gene encoding a Δ9elongase polypeptide is set forth in SEQ ID NO:26 (wherein at least 98codons have been optimized for expression in Yarrowia relative to SEQ IDNO:11).

Since naturally produced PUFAs in oleaginous yeast are limited to 18:2fatty acids (i.e., LA), and less commonly, 18:3 fatty acids (i.e., ALA),the oleaginous yeast will be genetically engineered to express multipleenzymes necessary for long-chain PUFA biosynthesis (thereby enablingproduction of e.g., ARA, EPA, DPA and DHA), in addition to the Δ9elongases described herein.

Specifically, in one embodiment this invention concerns an oleaginousyeast comprising:

-   -   (a) a first recombinant DNA construct comprising an isolated        polynucleotide encoding a Δ9 elongase polypeptide, operably        linked to at least one regulatory sequence; and,    -   (b) at least one additional recombinant DNA construct comprising        an isolated polynucleotide, operably linked to at least one        regulatory sequence, encoding a polypeptide selected from the        group consisting of: a Δ4 desaturase, a Δ5 desaturase, Δ6        desaturase, a Δ9 desaturase, a Δ12 desaturase, a Δ15 desaturase,        a Δ17 desaturase, a Δ8 desaturase, a C_(14/16) elongase, a        C_(16/18) elongase, a C_(18/20) elongase and a C_(20/22)        elongase.

In particularly preferred embodiments, the at least one additionalrecombinant DNA construct encodes a polypeptide having Δ8 desaturaseactivity.

Metabolic Engineering of Omega-3 and/or Omega-6 Fatty Acid Biosynthesisin Microbes

Knowledge of the sequences of the present Δ9 elongases will be usefulfor manipulating ω-3 and/or ω-6 fatty acid biosynthesis in various hostcells. Methods for manipulating biochemical pathways are well known tothose skilled in the art; and, it is expected that numerousmanipulations will be possible to maximize ω-3 and/or ω-6 fatty acidbiosynthesis in oleaginous yeasts, and particularly, in Yarrowialipolytica. This manipulation may require metabolic engineering directlywithin the PUFA biosynthetic pathway or additional manipulation ofpathways that contribute carbon to the PUFA biosynthetic pathway.Methods useful for up-regulating desirable biochemical pathways anddown-regulating undesirable biochemical pathways are well known to thoseskilled in the art.

For example, biochemical pathways competing with the ω-3 and/or ω-6fatty acid biosynthetic pathways for energy or carbon, or native PUFAbiosynthetic pathway enzymes that interfere with production of aparticular PUFA end-product, may be eliminated by gene disruption ordown-regulated by other means (e.g., antisense mRNA).

Detailed discussion of manipulations within the PUFA biosyntheticpathway as a means to increase ARA, EPA or DHA (and associatedtechniques thereof) are presented in U.S. Patent Publication No.2006-0094092-A1, U.S. Patent Publication No. 2006-0115881-A1 and U.S.Patent Publication No. 2006-0110806-A1, respectively, as are desirablemanipulations in the TAG biosynthetic pathway and the TAG degradationpathway (and associated techniques thereof).

Within the context of the present invention, it may be useful tomodulate the expression of the fatty acid biosynthetic pathway by anyone of the strategies described above. For example, the presentinvention provides methods whereby genes encoding key enzymes in the Δ9elongase/Δ8 desaturase biosynthetic pathway are introduced intooleaginous yeasts for the production of ω-3 and/or ω-6 fatty acids. Itwill be particularly useful to express the present the Δ9 elongase genesin oleaginous yeasts that do not naturally possess ω-3 and/or ω-6 fattyacid biosynthetic pathways and coordinate the expression of these genes,to maximize production of preferred PUFA products using various meansfor metabolic engineering of the host organism.

Microbial Fermentation Processes for PUFA Production

The transformed microbial host cell is grown under conditions thatoptimize expression of chimeric desaturase and elongase genes andproduce the greatest and most economical yield of desired PUFAs. Ingeneral, media conditions that may be optimized include the type andamount of carbon source, the type and amount of nitrogen source, thecarbon-to-nitrogen ratio, the amount of different mineral ions, theoxygen level, growth temperature, pH, length of the biomass productionphase, length of the oil accumulation phase and the time and method ofcell harvest. Microorganisms of interest, such as oleaginous yeast(e.g., Yarrowia lipolytica) are generally grown in complex media (e.g.,yeast extract-peptone-dextrose broth (YPD)) or a defined minimal mediathat lacks a component necessary for growth and thereby forces selectionof the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources are taught in U.S. Pat. No.7,238,482. Although it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing sources, preferred carbon sources are sugars (e.g.,glucose), glycerol, and/or fatty acids.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic(e.g., urea or glutamate) source. In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the oleaginoushost and promotion of the enzymatic pathways necessary for PUFAproduction. Particular attention is given to several metal ions (e.g.,Fe⁺², Cu⁺², Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids andPUFAs (Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle andR. Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of the transformant hostcells will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of PUFAs in oleaginous yeast (e.g., Yarrowia lipolytica).This approach is described in U.S. Pat. No. 7,238,482, as are varioussuitable fermentation process designs (i.e., batch, fed-batch andcontinuous) and considerations during growth.

Purification and Processing of PUFA Oils

PUFAs may be found in the host microorganisms as free fatty acids or inesterified forms such as acylglycerols, phospholipids, sulfolipids orglycolipids, and may be extracted from the host cells through a varietyof means well-known in the art. One review of extraction techniques,quality analysis and acceptability standards for yeast lipids is that ofZ. Jacobs (Critical Reviews in Biotechnology, 12(5/6):463-491 (1992)). Abrief review of downstream processing is also available by A. Singh andO. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).

In general, means for the purification of PUFAs may include extraction(e.g., U.S. Pat. No. 6,797,303 and U.S. Pat. No. 5,648,564) with organicsolvents, sonication, supercritical fluid extraction (e.g., using carbondioxide), saponification and physical means such as presses, orcombinations thereof. One is referred to the teachings of U.S. Pat. No.7,238,482 for additional details.

PUFA-Containing Oils for use in Foodstuffs, Health Food Products,Pharmaceuticals and Animal Feeds

The market place currently supports a large variety of food and feedproducts, incorporating ω-3 and/or ω-6 fatty acids (particularly e.g.,ALA, GLA, ARA, EPA, DPA and DHA). It is contemplated that the microbialbiomass comprising long-chain PUFAs, partially purified microbialbiomass comprising PUFAs, purified microbial oil comprising PUFAs,and/or purified PUFAs will function in food and feed products to impartthe health benefits of current formulations. More specifically, oils ofthe invention containing ω-3 and/or ω-6 fatty acids will be suitable foruse in a variety of food and feed products including, but not limitedto: food analogs, meat products, cereal products, baked foods, snackfoods and dairy products (see Patent Publication No. US-2006-0094092 fordetails).

Additionally, the present compositions may be used in formulations toimpart health benefit in medical foods including medical nutritionals,dietary supplements, infant formula as well as pharmaceutical products.One of skill in the art of food processing and food formulation willunderstand how the amount and composition of the present oils may beadded to the food or feed product. Such an amount will be referred toherein as an “effective” amount and will depend on the food or feedproduct, the diet that the product is intended to supplement or themedical condition that the medical food or medical nutritional isintended to correct or treat.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1.) Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofmicrobial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, 2^(nd) ed., Sinauer Associates: Sunderland,Mass. (1989). All reagents, restriction enzymes and materials used forthe growth and maintenance of microbial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified. E. coli strains were typically grownat 37° C. on Luria Bertani (LB) plates.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). DNA sequence was generated on an ABI Automaticsequencer using dye terminator technology (U.S. Pat. No. 5,366,860; EP272,007) using a combination of vector and insert-specific primers.Sequence editing was performed in Sequencher (Gene Codes Corporation,Ann Arbor, Mich.). All sequences represent coverage at least two timesin both directions. Comparisons of genetic sequences were accomplishedusing DNASTAR software (DNASTAR Inc., Madison, Wis.).

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” or “hr” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

Nomenclature for Expression Cassettes:

The structure of an expression cassette will be represented by a simplenotation system of “X::Y::Z”, wherein X describes the promoter fragment,Y describes the gene fragment, and Z describes the terminator fragment,which are all operably linked to one another.

Transformation and Cultivation of Yarrowia lipolytica:

Yarrowia lipolytica strains with ATCC Accession Nos. #20362, #76982 and#90812 were purchased from the American Type Culture Collection(Rockville, Md.). Yarrowia lipolytica strains were typically grown at28-30° C. in several media, according to the recipes shown below. Agarplates were prepared as required by addition of 20 g/L agar to eachliquid media, according to standard methodology.

YPD Agar Medium (Per Liter):

10 g of yeast extract [Difco], 20 g of Bacto peptone [Difco]; and 20 gof glucose.

Basic Minimal Media (MM) (Per Liter):

20 g glucose; 1.7 g yeast nitrogen base without amino acids; 1.0 gproline; and pH 6.1 (not adjusted).

Minimal Media+5-Fluoroorotic Acid (MM+5-FOA) (Per Liter):

20 g glucose, 6.7 g Yeast Nitrogen base, 75 mg uracil, 75 mg uridine andappropriate amount of FOA (Zymo Research Corp., Orange, Calif.), basedon FOA activity testing against a range of concentrations from 100 mg/Lto 1000 mg/L (since variation occurs within each batch received from thesupplier).

Transformation of Yarrowia lipolytica was performed according to themethod of Chen, D. C. et al. (Appl. Microbiol. Biotechnol.,48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia wasstreaked onto a YPD plate and grown at 30° C. for approximately 18 h.Several large loopfuls of cells were scraped from the plate andresuspended in 1 mL of transformation buffer, comprising: 2.25 mL of 50%PEG, average MW 3350; 0.125 mL of 2 M lithium acetate, pH 6.0; 0.125 mLof 2 M DTT; and (optionally) 50 μg sheared salmon sperm DNA. Then,approximately 500 ng of linear DNA (preferably comprising at least onechimeric gene) (or 100 ng circular plasmid) was incubated in 100 μL ofresuspended cells, and maintained at 39° C. for 1 h with vortex mixingat 15 min intervals. The cells were plated onto selection media platesand maintained at 30° C. for 2 to 3 days.

Fatty Acid Analysis of Yarrowia lipolytica:

For fatty acid analysis, cells were collected by centrifugation andlipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can.J. Biochem. Physiol., 37:911-917 (1959)). Fatty acid methyl esters wereprepared by transesterification of the lipid extract with sodiummethoxide (Roughan, G. and Nishida I., Arch Biochem Biophys.,276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30 m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μL of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μL hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Example 1 Synthesis of a cDNA Library from Euglena anabaena UTEX 373

The present Example describes the synthesis of a cDNA library fromEuglena anabaena UTEX 373. This work included the preparation of RNA,synthesis of cDNA, and generation of a cDNA library.

Growth of Euglena anabaena UTEX 373 and Preparation of RNA

Euglena anabaena UTEX 373 was obtained from Dr. Richard Triemer's lab atMichigan State University (East Lansing, Mich.). Approximately 2 mL ofculture was removed for lipid analysis and centrifuged at 1,800×g for 5min. The pellet was washed once with water and re-centrifuged. Theresulting pellet was dried for 5 min under vacuum, resuspended in 100 μLof trimethylsulfonium hydroxide (TMSH) and incubated at room temperaturefor 15 min with shaking. After incubation, 0.5 mL of hexane was addedand the vials were further incubated for 15 min at room temperature withshaking. Fatty acid methyl esters (5 μL injected from hexane layer) wereseparated and quantified using a Hewlett-Packard 6890 Gas Chromatographfitted with an Omegawax 320 fused silica capillary column (Supelco Inc.,Catalog No. 24152). The oven temperature was programmed to hold at 170°C. for 1.0 min, increase to 240° C. at 5° C./min and then hold for anadditional 1.0 min. Carrier gas was supplied by a Whatman hydrogengenerator. Retention times were compared to those for methyl esters ofstandards commercially available (Nu-Chek Prep, Inc. Catalog No. U-99-A)and the resulting chromatogram is shown in FIG. 2. The presence of EDA,ETrA, EPA and DHA in the fatty acid profile, with the absence of GLA andSTA, suggested that Euglena anabaena uses the Δ9 elongase/Δ8 desaturasepathway for long-chain (LC) PUFA biosynthesis and would be a good sourcefor LC-PUFA biosynthetic genes such as, but not limited to, Δ9elongases.

The remaining 5 mL of an actively growing culture was transferred into25 mL of AF-6 Medium (Watanabe & Hiroki, NIES-Collection List ofStrains, 5^(th) ed., National Institute for Environmental Studies,Tsukuba, 127 pp (2004)) in a 125 mL glass flask. Euglena anabaenacultures were grown at 22° C. with a 16 h light, 8 h dark cycle for 2weeks with very gentle agitation.

After 2 weeks, the culture (25 mL) was transferred to 100 mL of AF-6medium in a 500 mL glass bottle and the culture was grown for 1 month asdescribed above. After this time, two 50 mL aliquots were transferredinto two separate 500 mL glass bottles containing 250 mL of AF-6 mediumand the cultures were grown for two months as described above (giving atotal of ˜600 mL of culture). Next, the cultures were pelleted bycentrifugation at 1,800×g for 10 min, washed once with water andre-centrifuged. Total RNA was extracted from one of the resultingpellets using the RNA STAT-60™ reagent (TEL-TEST, Inc., Friendswood,Tex.) and following the manufacturer's protocol provided (use 5 mL ofreagent, dissolved RNA in 0.5 mL of water). In this way, 340 μg of totalRNA (680 μg/mL) was obtained from the pellet. The remaining pellet wasfrozen in liquid nitrogen and stored at −80° C. The mRNA was isolatedfrom all 340 μg of total RNA using the mRNA Purification Kit (AmershamBiosciences, Piscataway, N.J.) following the manufacturer's protocolprovided. In this way, 9.0 μg of mRNA was obtained.

Preparation of Euglena anabaena cDNA and Generation of cDNA Libraryeug1c

A cDNA library was generated using the Cloneminer™ cDNA LibraryConstruction Kit (Catalog No. 18249-029, Invitrogen Corporation,Carlsbad, Calif.) and following the manufacturer's protocol provided(Version B, 25-0608). Using the non-radiolabeling method, cDNA wassynthesized from 5.12 μg of mRNA (described above) using theBiotin-attB2-Oligo(dT) primer. After synthesis of the first and secondstrand, the attB1 adapter was added, ligated and the cDNA was sizefractionated using column chromatography. DNA from fractions wereconcentrated, recombined into pDONR™222 and transformed into E. coliElectroMAX™ DH10B™ T1 Phage-Resistant cells (Invitrogen Corporation).The Euglena anabaena library was named eug1c.

The cDNA library eug1c was plated onto LB+Kanamycin plates (approx.100,000 colonies), the colonies were scraped off and DNA was isolatedusing the QIAprep® Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.)following the manufacturer's protocol. In this way, a plasmid DNAsub-library from eug1c was obtained.

Example 2 Isolation of Full-Length Δ9 Elongases from Euglena anabaenaUTEX 373

The present Example describes the identification of cDNAs (SEQ ID NOs:1and 2) encoding Δ9 elongases from Euglena anabaena UTEX 373. This workincluded the generation of a probe derived from the Euglena gracilis Δ9elongase (EgD9e; SEQ ID NO:3) and the hybridization of the probe to thecDNA library eug1c in order to identify Δ9 elongase homologs fromEuglena anabaena UTEX 373.

Euglena gracilis Δ9 Elongase (EgD9e)

A clone from the Euglena cDNA library (eeg1c), called eeg1c.pk001.n5f,containing the Euglena gracilis Δ9 elongase (EgD9e; SEQ ID NO:3; whichis described in U.S. application Ser. No. 11/601,563 was used astemplate to amplify EgD9e with oligonucleotide primers oEugEL1-1 (SEQ IDNO:5) and oEugEL1-2 (SEQ ID NO:6) using the VentR® DNA Polymerase(Catalog No. M0254S, New England Biolabs Inc., Beverly, Mass.) followingthe manufacturer's protocol. The resulting DNA fragment was cloned intothe pCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol, toproduce pKR906 (SEQ ID NO:15).

Colony Lifts

Approximately 17,000 clones of Euglena anabaena cDNA library eug1c wereplated onto three large square (24 cm×24 cm) petri plates (Corning,Corning, N.Y.) each containing LB+50 μg/mL kanamycin agar media. Cellswere grown overnight at 37° C. and plates were then cooled to roomtemperature.

Biodyne B 0.45 μm membrane (Catalog No. 60207, Pall Corporation,Pensacola, Fla.) was trimmed to approximately 22 cm×22 cm and themembrane was carefully layed on top of the agar to avoid air bubbles.After incubation for 2 min at room temperature, the membrane was markedfor orientation, lifted off with tweezers and placed colony-side up onfilter paper soaked with 0.5 M sodium hydroxide and 1.5 M sodiumchloride. After denaturation for 4 min, the sodium hydroxide wasneutralized by placing the membrane on filter paper soaked with 0.5 MTris-HCL (pH 7.5) and 1.5 M sodium chloride for 4 min. This step wasrepeated and the membrane was rinsed briefly in 2×SSC buffer (20×SSC is3 M sodium chloride, 0.3 M sodium citrate; pH 7.0) and air dried onfilter paper.

Hybridization

Membranes were pre-hybridized at 65° C. in 200 mL hybridization solutionfor 2 hr. Hybridization solution contained 6×SSPE (20×SSPE is 3 M sodiumchloride, 0.2 M sodium phosphate, 20 mM EDTA; pH 7.4), 5×Denhardt'sreagent (100×Denhardt's reagent is 2%(w/v) Ficoll, 2% (w/v)polyvinylpyrrolidone, 2% (w/v) acetylated bovine serum albumin), 0.5%sodium dodecyl sulfate (SDS), 100 μg/mL sheared salmon sperm DNA and 5%dextran sulfate.

A DNA probe was made using an agarose gel purified NcoI/NotI DNAfragment, containing the Euglena gracilis Δ9 elongase gene, from pKR906(SEQ ID NO:15) labeled with P³² dCTP using the RadPrime DNA LabelingSystem (Catalog No. 18428-011, Invitrogen, Carlsbad, Calif.) followingthe manufacturer's instructions. Unincorporated P³² dCTP was separatedusing a NICK column (Catalog No. 17-0855-02, Amersham Biosciences,Piscataway, N.J.) following the manufacturer's instructions. The probewas denatured for 5 min at 100° C., placed on ice for 3 min and half wasadded to the hybridization solution.

The membrane was hybridized with the probe overnight at 65° C. withgentle shaking and then washed the following day twice with 2×SSCcontaining 0.5% SDS (5 min each) and twice with 0.2×SSC containing 0.1%SDS (15 min each). After washing, hyperfilm (Catalog No. RPN30K,Amersham Biosciences, Piscataway, N.J.) was exposed to the membraneovernight at −80° C.

Based on alignment of plates with the exposed hyperfilm, positivecolonies were picked using the blunt end of a Pasteur pipette into 1 mLof water and vortexed. Several dilutions were made and plated onto smallround Petri dishes (82 mm) containing LB media plus 50 μg/mL kanamycinto obtain around 100 well isolated colonies on a single plate. Liftswere done as described above except NytranN membrane circles (CatalogNo. 10416116, Schleicher & Schuell, Keene, N.H.) were used andhybridization was carried out in 100 mL using the remaining radiolabeledprobe. In this way, positive clones were confirmed.

Individual positive clones were grown at 37° C. in LB+50 μg/mL kanamycinliquid media and plasmid was purified using the QIAprep® Spin MiniprepKit (Qiagen Inc.) following the manufacturer's protocol.

DNA inserts were end-sequenced in 384-well plates, using vector-primedM13F universal primer (SEQ ID NO:7), M13rev-28 primer (SEQ ID NO:8) andthe poly(A) tail-primed WobbleT oligonucleotides, with the ABI BigDyeversion 3 Prism sequencing kit. For the sequencing reaction, 100-200 ngof template and 6.4 pmol of primer were used, and the following reactionconditions were repeated 25 times: 96° C. for 10 sec, 50° C. for 5 secand 60° C. for 4 min. After ethanol-based cleanup, cycle sequencingreaction products were resolved and detected on Perkin-Elmer ABI 3700automated sequencers. The WobbleT primer is an equimolar mix of 21 merpoly(T)A, poly(T)C, and poly(T)G, used to sequence the 3′ end of cDNAclones.

Sequences were aligned and compared using Sequencher™ (Version 4.2, GeneCodes Corporation, Ann Arbor, Mich.) and in this way, the clones couldbe categorized into one of two distinct groups based on insert sequence(designated as EaD9Elo1 and EaD9Elo2). Representative clones containingthe cDNA for each class of sequence were chosen for further study andsequences for each representative plasmid (i.e., pLF121-1 and pLF121-2)are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively. The sequenceshown by a string of NNNN's represents a region of the polyA tail whichwas not sequenced. The coding sequences for EaD9Elo1 and EaD9Elo2 areshown in SEQ ID NO:11 and SEQ ID NO:12, respectively. The correspondingamino acid sequences for EaD9Elo1 and EaD9Elo2 are shown in SEQ ID NO:13and SEQ ID NO:14, respectively.

Example 3 Primary Sequence Analysis of the Δ9 Elongase Sequences ofEuglena anabaena UTEX 373 (EaD9Elo1 and EaD9Elo2) and Comparison toOther Published Δ9 Elongase Sequences

The amino acid sequences for EaD9Elo1 (SEQ ID NO:13) and EaD9Elo2 (SEQID NO:14) were compared using the Clustal V method (Higgins, D. G. andSharp, P. M., Comput. Appl. Biosci., 5:151-153 (1989); Higgins et al.,Comput. Appl. Biosci., 8:189-191 (1992)) using the MegAlign™ v6.1program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.) with the default parameters for pairwise alignment(KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5 and GAP LENGTHPENALTY=10).

Compared to EaD9Elo1 (SEQ ID NO:13), EaD9Elo2 (SEQ ID NO:14) has 1 aminoacid substitution (i.e., R254Q; based on numbering for EaD9Elo1). Thenucleotide sequences of EaD9Elo1 (SEQ ID NO:11) and EaD9Elo2 (SEQ IDNO:12) differ by six base pairs over the full 774 bp lengths.

The amino acid sequences for EaD9Elo1 (SEQ ID NO:13) and EaD9Elo2 (SEQID NO:14) were evaluated by BLASTP (Basic Local Alignment Search Tool;Altschul et al., J. Mol. Biol., 215:403-410 (1993)) searches forsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL and DDBJdatabases) using default parameters with the filter turned off. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Both sequences yielded a pLog value of 38.70 (P value of 2e-39) versusthe Isochrysis galbana long chain polyunsaturated fatty acid elongationenzyme (IgD9e; SEQ ID NO:16) (NCBI Accession No. AAL37626(GI 17226123),locus AAL37626, CDS AF390174; Qi et al., FEBS Lett., 510:159-165 (2002))when compared to the “nr” database. BLAST scores and probabilitiesindicate that the instant nucleic acid fragments encode entire Euglenaanabaena Δ9 fatty acid elongases.

The amino acid sequences for EaD9Elo1 (SEQ ID NO:13) and EaD9Elo2 (SEQID NO:14) were compared to IgD9e (SEQ ID NO:16) and the Euglena gracilisΔ9 elongase amino acid sequence (EgD9e; SEQ ID NO:4; PCT Publication No.WO 2007/061845) using BlastP, Clustal V and the Jotun Hein methods ofsequence comparison. The % identity against IgD9e and EgD9e using eachmethod is shown in Table 4 and Table 5, respectively.

Sequence percent identity calculations were performed by the BlastP andClustal V methods, as described above. Sequence percent identitycalculations performed by the Jotun Hein method (Hein, J. J., Meth. Enz,183:626-645 (1990)) were done using the MegAlign™ v6.1 program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.)with the default parameters for pairwise alignment (KTUPLE=2).

TABLE 4 Sequence Comparison Of EaD9Elo1 (SEQ ID NO: 13) And Ead9elo2(SEQ ID NO: 14) To IgD9e (SEQ ID NO: 16) % Identity % Identity to %Identity to to IgD9e IgD9e by the IgD9e by the Desaturase by BLASTPJotun Hein Method Clustal V Method EaD9Elo1 37% 40.4% 32.9% EaD9Elo2 37%41.2% 32.9%

TABLE 5 Sequence Comparison Of EaD9Elo1 (SEQ ID NO: 13) And EaD9Elo2(SEQ ID NO: 14) To EgD9e (SEQ ID NO: 4) % Identity % Identity to %Identity to to EgD9e EgD9e by the EgD9e by the Desaturase by BLASTPJotun Hein Method Clustal V Method EaD9Elo1 77% 77.2% 77.1% EaD9Elo2 77%77.2% 77.1%

Example 4 Functional Analysis of the Euglena anabaena UTEX 373 Δ9Elongases in Yarrowia lipolytica

The present Example describes functional analysis of EaD9Elo1 (SEQ IDNO:13) and EaD9Elo2 (SEQ ID NO:14) in Yarrowia lipolytica. This workincluded the following steps: (1) Construction of Gateway®-compatibleYarrowia expression vector pY159; (2) transfer of EaD9Elo1 and EaD9Elo2into pY159 to produce pY173 and pY174; and, (3) comparison of lipidprofiles within transformant organisms comprising pY173 and pY174.

Construction of Gateway®-Compatible Yarrowia Expression Vector pY159

Plasmid pY5-30 (which was previously described in U.S. Pat. No.7,259,255), is a shuttle plasmid that can replicate both in E. coli andYarrowia lipolytica. Plasmid pY5-30 contains the following: a Yarrowiaautonomous replication sequence (ARS18); a ColE1 plasmid origin ofreplication; an ampicillin-resistance gene (Amp^(R)), for selection inE. coli; a Yarrowia LEU2 gene, for selection in Yarrowia; and a chimericTEF::GUS::XPR gene. Plasmid pDMW263 (SEQ ID NO:17) was created frompY5-30, by replacing the TEF promoter with the Yarrowia lipolyticaFBAINm promoter (U.S. Pat. No. 7,202,356) using techniques well known toone skilled in the art. Briefly, this promoter refers to a modifiedpromoter which is located in the 5′ upstream untranslated region infront of the ‘ATG’ translation initiation codon of thefructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by thefba1 gene and that is necessary for expression, plus a portion of 5′coding region that has an intron, wherein FBAINm has a 52 bp deletionbetween the ATG translation initiation codon and the intron of the FBAINpromoter (thereby including only 22 amino acids of the N-terminus) and anew translation consensus motif after the intron. Table 6 summarizes thecomponents of pDMW263 (SEQ ID NO:17).

TABLE 6 Components Of Plasmid pDMW263 (SEQ ID NO: 17) RE Sites andNucleotides Within Description of Fragment and SEQ ID NO: 17 ChimericGene Components 4992-4296 ARS18 sequence (GenBank Accession No. A17608)SaII/SacII FBAINm::GUS::XPR, comprising: (8505-2014) FBAINm: Yarrowialipolytica FBAINm promoter (U.S. Pat. No. 7,202,356); GUS: E. coli geneencoding β-glucuronidase (Jefferson, R. A. Nature, 14: 342: 837-838(1989); XPR: ~100 bp of the 3′ region of the Yarrowia Xpr gene (GenBankAccession No. M17741) 6303-8505 Yarrowia Leu2 gene (GenBank AccessionNo. AF260230)

The NcoI/SalI DNA fragment from pDMW263 (SEQ ID NO:17), containing theYarrowia lipolytica FBAINm promoter, was cloned into the NcoI/SalI DNAfragment of pDMW237 (SEQ ID NO:18), previously described in PCTPublication No. WO 2006/012325 (the contents of which are herebyincorporated by reference), containing a synthetic Δ9 elongase genederived from Isochrysis galbana and codon-optimized for expression inYarrowia lipolytica (IgD9eS), to produce pY115 (SEQ ID NO:19; FIG. 3A).In FIG. 3A, the modified FBAINm promoter is labeled as FBA1+Intron,while it is labeled as YAR FBA1 PRO+Intron in FIGS. 3B, 3C and 3D.

The FBAINm promoter was amplified from plasmid pY115 (SEQ ID NO:19),using PCR with oligonucleotide primers oYFBA1 (SEQ ID NO:20) andoYFBA1-6 (SEQ ID NO:21). Primer oYFBA1 (SEQ ID NO:20) was designed tointroduce a Bg/II site at the 5′ end of the promoter and primer oYFBA1-6(SEQ ID NO:21) was designed to introduce a NotI site at the 3′ end ofthe promoter while removing the NcoI site and thus, the ATG start codon.The resulting PCR fragment was digested with Bg/II and NotI and clonedinto the Bg/II/NotI fragment of pY115, containing the vector backbone,to form pY158 (SEQ ID NO:22).

Plasmid pY158 (SEQ ID NO:22) was digested with NotI and the resultingDNA ends were filled. After filling to form blunt ends, the DNAfragments were treated with calf intestinal alkaline phosphatase andseparated using agarose gel electrophoresis. The 6992 bp fragmentcontaining the Yarrowia lipolytica FBAINm promoter was excised from theagarose gel and purified using the QIAquick® Gel Extraction Kit (QiagenInc., Valencia, Calif.) following the manufacturer's protocol. Thepurified 6992 bp fragment was ligated with cassette rfA using theGateway Vector Conversion System (Catalog No. 11823-029, InvitrogenCorporation) following the manufacturer's protocol to form Yarrowialipolytica Gateway® destination vector pY159 (SEQ ID NO:23; FIG. 3B).

Construction of Yarrowia Expression Vectors pY173 and pY174

Using the Gateway® LR Clonase™ II enzyme mix (Catalog No. 11791-020,Invitrogen Corporation) and following the manufacturer's protocol, thecDNA inserts from pLF121-1 (SEQ ID NO:9; Example 2) and pLF121-2 (SEQ IDNO:10; Example 2) were transferred to pY159 (SEQ ID NO:23) to form pY173(SEQ ID NO:24; FIG. 3C) and pY174 (SEQ ID NO:25; FIG. 3D), respectively.

Functional Analysis of EaD9Elo1 And EaD9Elo2 in Yarrowia lipolyticaStrain Y2224

Strain Y2224 was isolated in the following manner: Yarrowia lipolyticaATCC #20362 cells from a YPD agar plate (1% yeast extract, 2%bactopeptone, 2% glucose, 2% agar) were streaked onto a MM plate (75mg/L each of uracil and uridine, 6.7 g/L YNB with ammonia sulfate,without amino acid, and 20 g/L glucose) containing 250 mg/L 5-FOA (ZymoResearch). Plates were incubated at 28° C. and four of the resultingcolonies were patched separately onto MM plates containing 200 mg/mL5-FOA and MM plates lacking uracil and uridine to confirm uracil Ura3auxotrophy.

Strain Y2224 was transformed with pY173 (SEQ ID NO:24; FIG. 3C) andpY174 (SEQ ID NO:25; FIG. 3D) as described in the General Methods.

Single colonies of transformant Yarrowia lipolytica containing pY173 andpY174 were grown in 3 mL MM lacking uracil at 30° C. for 16 h afterwhich cells were centrifuged at 250 rpm to pellet. Cells were washedonce with water, pelleted by centrifugation and air dried. Pellets weretransesterified (Roughan, G. and Nishida, I., Arch. Biochem. Biophys.,276(1):38-46 (1990)) with 500 μL of 1% sodium methoxide for 30 min at50° C. after which 500 μL of 1 M sodium chloride and 100 μL of heptanewere added. After thorough mixing and centrifugation, fatty acid methylesters (FAMEs) were analyzed by GC. FAMEs (5 μL injected from hexanelayer) were separated and quantified using a Hewlett-Packard 6890 GasChromatograph fitted with an Omegawax 320 fused silica capillary column(Catalog No. 24152, Supelco Inc.). The oven temperature was programmedto hold at 220° C. for 2.6 min, increase to 240° C. at 20° C./min andthen hold for an additional 2.4 min. Carrier gas was supplied by aWhatman hydrogen generator. Retention times were compared to those formethyl esters of standards commercially available (Nu-Chek Prep, Inc.).

The fatty acid profiles for Yarrowia lipolytica expressing pY173 andpY174 are shown in Table 7. Fatty acids are identified as 16:0, 16:1,18:0, 18:1 (oleic acid), LA, 20:0, 20:1(11), EDA, 22:0, 24:0 and 24:1.Percent Δ9 elongation (Δ9% Elong) was calculated by dividing the weight% (wt %) for EDA by the sum of the wt % for EDA and LA and multiplyingby 100 to express as a %. Average is indicated by Ave.

TABLE 7 Fatty Acid Composition (Wt %) For Yarrowia lipolytica ExpressingpY173 (EaD9Elo1) And pY174 (EaD9Elo2) 20:1 A9 Ave. Δ9 Event 16:0 16:118:0 18:1 LA 20:0 (11) EDA 22:0 24:0 24:1 % Elong % Elong PY173-1 16.714.5 4.1 46.5 12.5 0.2 0.2 3.6 0.2 1.4 0.1 22.2 22.7 pY173-2 16.6 14.24.1 46.8 12.4 0.2 0.2 3.7 0.2 1.5 0.1 22.7 pY173-3 16.5 14.0 4.2 47.112.3 0.2 0.2 3.7 0.2 1.5 0.2 23.2 pY174-1 16.9 14.3 4.2 46.8 12.5 0.20.2 3.2 0.2 1.4 0.1 20.5 21.1 pY174-2 17.0 14.1 4.3 47.4 11.8 0.2 0.23.3 0.2 1.4 0.1 21.6 pY174-3 17.0 14.2 4.3 47.2 11.9 0.2 0.2 3.2 0.2 1.40.2 21.2

Example 5 Synthesis of a Codon-Optimized Δ9 Elongase Gene for Yarrowialipolytica (EaD9ES)

The codon usage of the Δ9 elongase gene (EaD9Elo1) of Euglena anabaenawas optimized for expression in Yarrowia lipolytica, in a manner similarto that described in PCT Publication No. WO 2004/101753 and U.S. Pat.No. 7,125,672. Specifically, a codon-optimized Δ9 elongase gene(designated “EaD9ES”; SEQ ID NO:26) was designed based on the codingsequence of EaD9Elo1 (SEQ ID NO:11), according to the Yarrowia codonusage pattern (PCT Publication No. WO 2004/101753), the consensussequence around the ‘ATG’ translation initiation codon, and the generalrules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene,265(1-2):11-23 (2001)). In addition to modification of the translationinitiation site, 106 bp of the 774 bp coding region were modified(13.7%) and 98 codons were optimized (38.0%). The GC content (52.1%) wasabout the same between the wild type gene (i.e., EaD9Elo1) and thesynthetic gene (i.e., EaD9ES). A NcoI site and NotI sites wereincorporated around the translation initiation codon and after the stopcodon of EaD9ES (SEQ ID NO:26), respectively. FIGS. 4A and 4B show acomparison of the nucleotide sequences of EaD9Elo1 (SEQ ID NO:11) andEaD9ES (SEQ ID NO:26). The protein sequence encoded by thecodon-optimized gene (i.e., SEQ ID NO:27) is identical to that of thewildtype protein sequence (i.e., SEQ ID NO:13). The designed EaD9ES genewas synthesized by GenScript Corporation (Piscataway, N.J.) and clonedinto pUC57 (GenBank Accession No. Y14837) to generate pEaD9ES (SEQ IDNO:28; FIG. 5A).

Example 6 Construction and Functional Analysis of Yarrowia lipolyticaExpression Vector pZUFmEaD9ES, Comprising a Synthetic Δ9 Elongase Gene(Derived from Euglena anabaena), Codon-Optimized for Expression inYarrowia lipolytica (EaD9ES)

The present Example describes the functional expression of Yarrowialipolytica vector pZUFmEaD9ES, comprising a chimericFBAINm::EaD9ES::Pex20 gene, wherein EaD9ES is a synthetic Δ9 elongasederived from Euglena anabaena and codon-optimized for expression inYarrowia. The plasmid pZUFmEaD9ES (FIG. 5B) contained the followingcomponents:

TABLE 8 Components Of Plasmid pZUFmEaD9ES (SEQ ID NO: 29) RE Sites AndNucleotides Within Description Of Fragment And SEQ ID NO: 29 ChimericGene Components Swa I/BsiW I FBAINm::EaD9ES::Pex20, comprising:(6067-318)  FBAINm: Yarrowia lipolytica FBAIN promoter (U.S. Pat. No.7,202,356) EaD9ES: codon-optimized Δ9 elongase (SEQ ID NO: 26), derivedfrom Euglena anabaena Pex20: Pex20 terminator sequence of Yarrowia Pex20gene (GenBank Accession No. AF054613) 1354-474  ColE1 plasmid origin ofreplication 2284-1424 Ampicillin-resistance gene (AmpR) for selection inE. coli 3183-4487 Yarrowia autonomous replication sequence (ARS18;GenBank Accession No. A17608) 6031-4530 Yarrowia Ura 3 gene (GenBankAccession No. AJ306421)

Functional Analysis of Yarrowia lipolytica Transformants ComprisingpZUFmEaD9ES

Plasmid pZUFmEaD9ES was transformed into strain Y2224 (the FOA resistantmutant from an autonomous mutation of the Ura3 gene of wildtype Yarrowiastrain ATCC #20362), as described in the General Methods. Thetransformants were selected on MM plates. After 2 days growth at 30° C.,transformants were picked and re-streaked onto fresh MM plates. Oncegrown, these strains were individually inoculated into 3 mL liquid MM at30° C. and shaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed that there were about 2.2% C20:2 (EDA) and 15.3%C18:2 (LA) of total lipids produced in all 5 transformants, wherein theconversion efficiency of C18:2 to C20:2 in these 5 strains wasdetermined to be about 13%. Thus, this experimental data demonstratedthat the synthetic Euglena anabaena Δ9 elongase codon-optimized forexpression in Yarrowia lipolytica (i.e., EaD9ES, as set forth in SEQ IDNOs:26 and 27) actively elongates LA to EDA.

1. A method for the production of at least one polyunsaturated fattyacid selected from the group consisting of eicosadienoic acid andeicosatrienoic acid comprising: a) providing a transformed microbialhost cell comprising: (i) a recombinant nucleotide molecule encoding apolypeptide having delta-9 elongase activity, wherein the polypeptidehas at least 90% amino acid identity, based on the Clustal V method ofalignment, when compared to the amino acid sequence as set forth in SEQID NO:13 or SEQ ID NO:14; and (ii) a source of a substratepolyunsaturated fatty acid selected from the group consisting oflinoleic acid and alpha-linolenic acid; b) growing the transformedmicrobial host cell of step (a) under conditions wherein linoleic acidis converted to eicosadienoic acid and alpha-linolenic acid is convertedto eicosatrienoic acid; and c) optionally recovering the eicosadienoicacid and/or eicosatrienoic acid of step (b).
 2. The method of claim 1,wherein the transformed microbial host cell is a Yarrowia, saidpolypeptide has the full-length amino acid sequence set forth in SEQ IDNo. 27, and the recombinant nucleotide molecule comprises at least 98codons which are optimized for expression in Yarrowia.
 3. The methodaccording to claim 1, wherein: a) the recombinant nucleotide moleculehas the full-length nucleic acid sequence selected from the groupconsisting of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:26; and b) thehost cell is Yarrowia lipolytica.
 4. The method according to claim 1wherein the transformed microbial host cell is selected from the groupconsisting of yeast, algae, bacteria, euglenoids, stramenopiles andfungi.
 5. The method according to claim 4 wherein the transformedmicrobial host cell is a fungus of the genus Mortierella.
 6. The methodaccording to claim 4 wherein the transformed microbial host cell is astramenopile selected from the group consisting of: Thraustochytrium andSchizochytrium.
 7. The method according to claim 4 wherein thetransformed microbial host cell is an oleaginous yeast.
 8. The methodaccording to claim 7 wherein the transformed microbial host cell is anoleaginous yeast selected from the group consisting of: Yarrowia,Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon andLipomyces.